Compositions for the electronic detection of analytes utilizing monolayers

ABSTRACT

The present invention relates to the use of self-assembled monolayers with mixtures of conductive oligomers and insulators to detect target analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/452,277, filed Nov. 30, 1999, now U.S. Pat. No. 7,160,678, whichis a continuation application of U.S. application Ser. No. 08/911,085,filed Aug. 14, 1997, now U.S. Pat. No. 6,090,933, and is a continuationapplication of U.S. application Ser. No. 08/911,589, filed Aug. 14,1997, now U.S. Pat. No. 6,232,062, which claims the benefit under 35U.S.C. § 119(e) of U.S. application Ser. No. 60/049,489, filed Jun. 12,1997, and U.S. application Ser. No. 60/040,153, filed Mar. 7, 1997. SaidU.S. application Ser. No. 08/911,085, is a continuation application ofU.S. application Ser. No. 08/873,978, filed Jun. 12, 1997, now U.S. Pat.No. 7,014,992 which is a continuation application of U.S. applicationSer. No. 08/743,798, filed Nov. 5, 1996, now U.S. Pat. No. 6,096,273,which is a continuation application of U.S. application Ser. No.08/873,597, filed Jun. 12, 1997, which claims the benefit under 35U.S.C. § 365 of PCT/US97/20014, filed Nov. 5, 1997, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. application Ser. No.60/040,155, filed Mar. 7, 1997, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the use of self-assembled monolayerswith mixtures of conductive oligomers and insulators to detect targetanalytes.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance.

The use of self-assembled monolayers (SAMs) on surfaces for binding anddetection of biological molecules has recently been explored. See forexample WO98120162; PCT US98/12430; PCT US98/12082; PCT US99/01705; andU.S. Pat. No. 5,620,850; and references cited therein.

Accordingly, it is an object of the invention to provide novel methodsand compositions for the electronic detection of target analytes usingself-assembled monolayers.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compositions comprising electrodes comprising a monolayercomprising conductive oligomers, and a capture binding ligand. Thecomposition further comprises a recruitment linker that comprises atleast one covalently attached electron transfer moiety, and a solutionbinding ligand that will bind to a target analyte.

In a further embodiment, the invention provides methods of detecting atarget analyte in a test sample comprising attaching said target analyteto an electrode comprising a monolayer of conductive oligomers viabinding to a capture binding ligand. Recruitment linkers, or signalcarriers, are directly or indirectly attached to the target analyte toform an assay complex. The method further comprises detecting electrontransfer between said ETM and said electrode.

Kits and apparatus comprising the compositions of the method are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C depict three preferred embodiments for attaching atarget nucleic acid sequence to the electrode. FIG. 1A depicts a targetsequence 120 hybridized to a capture probe 100 linked via a attachmentlinker 106, which as outlined herein may be either a conductive oligomeror an insulator. The electrode 105 comprises a monolayer of passivationagent 107, which can comprise conductive oligomers (herein depicted as108) and/or insulators (herein depicted as 109), and preferably both. Asfor all the embodiments depicted in the figures, n is an integer of atleast 1, although as will be appreciated by those in the art, the systemmay not utilize a capture probe at all (i.e. n is zero), although thisis generally not preferred. The upper limit of n will depend on thelength of the target sequence and the required sensitivity. FIG. 1Bdepicts the use of a single capture extender probe 110 with a firstportion 111 that will hybridize to a first portion of the targetsequence 120 and a second portion that will hybridize to the captureprobe 100. FIG. 1C depicts the use of two capture extender probes 110and 130. The first capture extender probe 110 has a first portion 111that will hybridize to a first portion of the target sequence 120 and asecond portion 112 that will hybridize to a first portion 102 of thecapture probe 100. The second capture extender probe 130 has a firstportion 132 that will hybridize to a second portion of the targetsequence 120 and a second portion 131 that will hybridize to a secondportion 101 of the capture probe 100. As will be appreciated by those inthe art, while these systems depict nucleic acid targets, theseattachment configurations may be used with non-nucleic acid capturebinding ligands; see for example FIG. 2C.

FIGS. 2A, 2B, 2C and 2D depict several embodiments of the invention.FIG. 2A is directed to the use of a capture binding ligand 200 attachedvia an attachment linker 106 to the electrode 105. Target analyte 210binds to the capture binding ligand 200, and a solution binding ligand22 with a directly attached recruitment linker 230 with ETMs 135. FIG.2B depicts a similar embodiment using an indirectly attached recruitmentlinker 145 that binds to a second portion 240 of the solution bindingligand 220. FIG. 2C depicts the use of an anchor ligand 100 (referred toherein as an anchor probe when the ligand comprises nucleic acid) tobind the capture binding ligand 200 comprising a portion 120 that willbind to the anchor probe 100. As will be appreciated by those in theart, any of the FIG. 1 embodiments may be used here as well. FIG. 2Ddepicts the use of an amplifier probe 145. As will be appreciated bythose in the art, any of the FIG. 3 amplifier probe configurations maybe used here as well.

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G and 3H depict some of the embodimentsof the invention. While depicted for nucleic acids, they can be used innon-nucleic acid embodiments as well. All of the monolayers depictedherein show the presence of both conductive oligomers 108 and insulators107 in roughly a 1:1 ratio, although as discussed herein, a variety ofdifferent ratios may be used, or the insulator may be completely absent.In addition, as will be appreciated by those in the art, any one ofthese structures may be repeated for a particular target sequence; thatis, for long target sequences, there may be multiple assay complexesformed. Additionally, any of the electrode-attachment embodiments ofFIG. 3 may be used in any of these systems.

FIGS. 3A, 3B and 3D have the target sequence 120 containing the ETMs135; as discussed herein, these may be added enzymatically, for exampleduring a PCR reaction using nucleotides modified with ETMs, resulting inessentially random incorporation throughout the target sequence, oradded to the terminus of the target sequence. FIG. 3C depicts the use oftwo different capture probes 100 and 100′, that hybridize to differentportions of the target sequence 120. As will be appreciated by those inthe art, the 5′-3′ orientation of the two capture probes in thisembodiment is different.

FIG. 3C depicts the use of recruitment linkers (referred to herein aslabel probes when nucleic acids are used) 145 that hybridize directly tothe target sequence 120. FIG. 3C shows the use of a label probe 145,comprising a first portion 141 that hybridizes to a portion of thetarget sequence 120, a second portion 142 comprising ETMs 135.

FIGS. 3E, 3F and 3G depict systems utilizing label probes 145 that donot hybridize directly to the target, but rather to amplifier probes 150that are directly (FIG. 3E) or indirectly (FIGS. 3F and 3G) hybridizedto the target sequence. FIG. 3E utilizes an amplifier probe 150 has afirst portion 151 that hybridizes to the target sequence 120 and atleast one second portion 152, i.e. the amplifier sequence, thathybridizes to the first portion 141 of the label probe. FIG. 3F issimilar, except that a first label extender probe 160 is used,comprising a first portion 161 that hybridizes to the target sequence120 and a second portion 162 that hybridizes to a first portion 151 ofamplifier probe 150. A second portion 152 of the amplifier probe 150hybridizes to a first portion 141 of the label probe 140, which alsocomprises a recruitment linker 142 comprising ETMs 135. FIG. 3G adds asecond label extender probe 170, with a first portion 171 thathybridizes to a portion of the target sequence 120 and a second portionthat hybridizes to a portion of the amplifier probe.

FIG. 3H depicts a system that utilizes multiple label probes. The firstportion 141 of the label probe 140 can hybridize to all or part of therecruitment linker 142.

FIGS. 4A and 4B show two competitive type assays of the invention. FIG.4A utilizes the replacement of a target analyte 210 with a targetanalyte analog 310 comprising a directly attached recruitment linker145. As will be appreciated by those in the art, an indirectly attachedrecruitment linker can also be used, as shown in FIG. 2B. FIG. 4B showsa competitive assay wherein the target analyte 210 and the targetanalyte analog 310 attached to the surface compete for binding of asolution binding ligand 220 with a directly attached recruitment linker145 (again, an indirectly attached recruitment linker can also be used,as shown in FIG. 2B). In this case, a loss of signal may be seen.

FIGS. 5A, 513, 5C, 5D and 5E depict additional embodiments of theinvention. FIG. 5A shows a conformation wherein the addition of targetalters the conformation of the binding ligands, causing the recruitmentlinker 145 to be placed near the monolayer surface. FIG. 5B shows theuse of the present invention in candidate bioactive agent screening,wherein the addition of a drug candidate to target causes the solutionbinding ligand to dissociate, causing a loss of signal. In addition, thesolution binding ligand may be added to another surface and be bound, asis generally depicted in FIG. 5C for enzymes. FIG. 5C depicts the use ofan enzyme to cleave a substrate 260 comprising a recruitment linker 145,causing a loss of signal. The cleaved piece may also be added to anadditional electrode, causing an increase in signal. FIG. 5D shows theuse of two different capture binding ligands 200; these may also beattached to the electrode using capture extender ligands. FIG. 5E addsan additional ‘sandwich component’ in the form of an additional solutionbinding ligand 250.

FIGS. 6A-6R depict nucleic acid detection systems. FIGS. 6A and 6B havethe target sequence 5 containing the ETMs 6; as discussed herein, thesemay be added enzymatically, for example during a PCR reaction usingnucleotides modified with ETMs, resulting in essentially randomincorporation throughout the target sequence, or added to the terminusof the target sequence. FIG. 6A shows attachment of a capture probe 10to the electrode 20 via a linker 15, which as discussed herein can beeither a conductive oligomer 25 or an insulator 30. The target sequence5 contains ETMs 6. FIG. 6B depicts the use of a capture extender probe11, comprising a first portion 12 that hybridizes to a portion of thetarget sequence and a second portion 13 that hybridizes to the captureprobe 10.

FIG. 6C depicts the use of two different capture probes 10 and 10′, thathybridize to different portions of the target sequence 5. As will beappreciated by those in the art, the 5′-3′ orientation of the twocapture probes in this embodiment is different.

FIGS. 6D to 6H depict the use of label probes 40 that hybridize directlyto the target sequence 5. FIG. 6D shows the use of a label probe 40,comprising a first portion 41 that hybridizes to a portion of the targetsequence 5, a second portion 42 that hybridizes to the capture probe 10and a recruitment linker 50 comprising ETMs 6. A similar embodiment isshown in FIG. 6E, where the label probe 40 has an additional recruitmentlinker 50. FIG. 6F depicts a label probe 40 comprising a first portion41 that hybridizes to a portion of the target sequence 5 and arecruitment linker 50 with attached ETMs 6. The parentheses highlightthat for any particular target sequence 5 more than one label probe 40may be used, with n being an integer of at least 1. FIG. 6G depicts theuse of the FIG. 6E label probe structures but includes the use of asingle capture extender probe 11, with a first portion 12 thathybridizes to a portion of the target sequence and a second portion 13that hybridizes to the capture probe 10. FIG. 6H depicts the use of theFIG. 6F label probe structures but utilizes two capture extender probes11 and 16. The first capture extender probe 11 has a first portion 12that hybridizes to a portion of the target sequence 5 and a secondportion 13 that hybridizes to a first portion 14 of the capture probe10. The second capture extender probe 16 has a first portion 18 thathybridizes to a second portion of the target sequence 5 and a secondportion 17 that hybridizes to a second portion 19 of the capture probe10.

FIGS. 61, 6J and 6K depict systems utilizing label probes 40 that do nothybridize directly to the target, but rather to amplifier probes. Thusthe amplifier probe 60 has a first portion 65 that hybridizes to thetarget sequence 5 and at least one second portion 70, i.e. the amplifiersequence, that hybridizes to the first portion 41 of the label probe.

FIGS. 6L, 6M and 6N depict systems that utilize a first label extenderprobe 80. In these embodiments, the label extender probe 80 has a firstportion 81 that hybridizes to a portion of the target sequence 5, and asecond portion 82 that hybridizes to the first portion 65 of theamplifier probe 60.

FIG. 60 depicts the use of two label extender probes 80 and 90. Thefirst label extender probe 80 has a first portion 81 that hybridizes toa portion of the target sequence 5, and a second portion 82 thathybridizes to a first portion 62 of the amplifier probe 60. The secondlabel extender probe 90 has a first portion 91 that hybridizes to asecond portion of the target sequence 5 and a second portion 92 thathybridizes to a second portion 61 of the amplifier probe 60.

FIG. 6P depicts a system utilizing a label probe 40 hybridizing to theterminus of a target sequence 5.

FIGS. 6Q and 6R depict systems that utilizes multiple label probes. Thefirst portion 41 of the label probe 40 can hybridize to all (FIG. 6R) orpart (FIG. 6Q) of the recruitment linker 50.

FIG. 7 depicts the use of an activated carboxylate for the addition of anucleic acid functionalized with a primary amine to a pre-formed SAM.

FIG. 8 shows a representative hairpin structure. 500 is a target bindingsequence, 510 is a loop sequence, 520 is a self-complementary region,530 is substantially complementary to a detection probe, and 530 is the“sticky end”, that is, a portion that does not hybridize to any otherportion of the; probe, that contains the ETMs.

FIG. 9 depicts the synthesis of an adenosine comprising a ferrocenelinked to the ribose.

FIG. 10 depicts the synthesis of a “branch” point (in this case anadenosine), to allow the addition of ETM polymers.

FIG. 11 depicts the synthetic scheme of a preferred attachment of anETM, in this case ferrocene, to a nucleoside via the phosphate.

FIG. 12 depicts the synthetic scheme of ethylene glycol terminatedconductive oligomers.

FIG. 13 depicts the synthesis of an insulator to the ribose of anucleoside for attachment to an electrode.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G, 14H, 14I, 14J and 14K depict anumber of different embodiments of the invention; the results are shownin Example 7.

FIGS. 15A-150 depict a number of different compositions of theinvention; the results are shown in Example 7 and 8. FIG. 15A depicts I,also referred to as P290. FIG. 15B depicts II, also referred to as P291.FIG. 15C depicts III, also referred to as W31. FIG. 15D depicts IV, alsoreferred to as N6. FIG. 15E depicts V, also referred to as P292. FIG.51F depicts II, also referred to as C23. FIG. 15G depicts VII, alsoreferred to as C15. FIG. 15H depicts VIII, also referred to as C95. FIG.151 depicts Y63. FIG. IJ depicts another compound of the invention. FIG.15K depicts Nil. FIG. 15L depicts C131, with a phosphoramidite group anda DMT protecting group. FIG. 15M depicts W38, also with aphosphoramidite group and a DMT protecting group. FIG. 15N depicts thecommercially available moiety that enables “branching” to occur, as itsincorporation into a growing oligonucleotide chain results in additionat both the DMT protected oxygens. FIG. 150 depicts glen, also with aphosphoramidite group and a DMT protecting group, that serves as anonnucleic acid linker. FIGS. 15A to 15G and 15J are shown without thephosphoramidite and protecting groups (i.e. DMT) that are readily added.

FIGS. 16A-16B depict representative scans from the experiments outlinedin Example 7. Unless otherwise noted, all scans were run at initialvoltage −0.11 V, final voltage 0.5 V, with points taken every 10 mV,amplitude of 0.025, frequency of 10 Hz, a sample period of I sec, aquiet time of 2 sec. FIG. 16A has a peak potential of 0.160 V, a peakcurrent of 1.092×10⁻⁸ A, and a peak A of 7.563×10⁻¹⁰ VA.

FIG. 17 depicts the synthetic scheme for a ribose linked ETM, W38.

FIGS. 18A and 18B depicts two phosphate attachments of conductiveoligomers that can be used to add the conductive oligomers at the 5′position, or any position.

FIG. 19 depicts a schematic of the synthesis of simultaneousincorporation of multiple ETMs into a nucleic acid, using a “branch”point nucleoside.

FIG. 20 depicts a schematic of an alternate method of adding largenumbers of ETMs simultaneously to a nucleic acid using a “branch” pointphosphoramidite, as is known in the art. As will be appreciated by thosein the art, each end point can contain any number of ETMs.

FIGS. 21A, 21B, 21C, 21D and 21E depict different possibleconfigurations of label probes and attachments of ETMs. In FIGS. 21A-C,the recruitment linker is nucleic acid; in FIGS. 21D and E, is not.A=nucleoside replacement; B=attachment to a base; C=attachment to aribose; D=attachment to a phosphate; E=metallocene polymer (although asdescribed herein, this can be a polymer of other ETMs as well), attachedto a base, ribose or phosphate (or other backbone analogs); F=dendrimerstructure, attached via a base, ribose or phosphate (or other backboneanalogs); G=attachment via a “branching” structure, through base, riboseor phosphate (or other backbone analogs); H=attachment of metallocene(or other ETM) polymers; I=attachment via a dendrimer structure;J=attachment using standard linkers.

FIGS. 22A and 22B depict some of the sequences used in the Examples (SEQID NOS:1-27).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the electronic detection ofanalytes. Previous work, described in PCT US97/20014, is directed to thedetection of nucleic acids, and utilizes nucleic acids covalentlyattached to electrodes using conductive oligomers, i.e. chemical“wires”. Upon formation of double stranded nucleic acids containingelectron transfer moieties (ETMs), electron transfer can proceed throughthe stacked n-orbitals of the heterocyclic bases to the electrode, thusenabling electronic detection of target nucleic acids. In the absence ofthe stacked n-orbitals, i.e. when the target strand is not present,electron transfer is negligible, thus allowing the use of the system asan assay. This previous work also reported on the use of self-assembledmonolayers (SAMs) to electronically shield the electrodes from solutioncomponents and significantly decrease the amount of non-specific bindingto the electrodes.

The present invention is directed to the discovery that present orabsence of ETMs can be directly detected on a surface of a monolayer ifthe monolayer comprises conductive oligomers, and preferably mixtures ofconductive oligomers and insulators. Thus, for example, when the targetanalyte is a nucleic acid, the electrons from the ETMs need not travelthrough the stacked n orbitals in order to generate a signal. Instead,the presence of ETMs on the surface of a SAM, that comprises conductiveoligomers, can be directly detected. Thus, upon binding of a targetanalyte to a binding species on the surface, a recruitment linkercomprising an ETM is brought to the surface, and detection of the ETMcan proceed. Thus, the role of the target analyte and binding species isto provide specificity for a recruitment of ETMs to the surface, wherethey can be detected using the electrode. Without being bound by theory,one possible mechanism is that the role of the SAM comprising theconductive oligomers is to “raise” the electronic surface of theelectrode, while still providing the benefits of shielding the electrodefrom solution components and reducing the amount of non-specific bindingto the electrodes.

The invention can be generally described as follows, with a number ofpossible embodiments depicted in the Figures. In a preferred embodiment,as depicted in FIG. 2, an electrode comprising a self-assembledmonolayer (SAM) of conductive oligomers, and preferably a mixture ofconductive oligomers and insulators, and a covalently attached targetanalyte binding ligand (frequently referred to herein as a “capturebinding ligand”) is made. The target analyte is added, which binds tothe support-bound binding ligand A solution binding ligand is added,which may be the same or different from the first binding ligand, whichcan also bind to the target analyte, forming a “sandwich” of sorts. Thesolution binding ligand either comprises a recruitment linker containingETMs, or comprises' a portion that will either directly or indirectlybind a recruitment linker containing the ETMs. This “recruitment” ofETMs to the surface of the monolayer allows electronic detection viaelectron transfer between the ETM and the electrode. In the absence ofthe target analyte, the recruitment linker is either washed away or notin sufficient proximity to the surface to allow detection.

In an alternate preferred embodiment, as depicted in FIG. 4, acompetitive binding type assay is run. In this embodiment, the targetanalyte in the sample is replaced by a target analyte analog as isdescribed below and generally known in the art. The analog comprises adirectly or indirectly attached recruitment linker comprising at leastone ETM. The binding of the analog to the capture binding ligandrecruits the ETM to the surface and allows detection based on electrontransfer between the ETM and the electrode.

In an additional preferred embodiment, as depicted in FIG. 4B, acompetitive assay wherein the target analyte and a target analyte analogattached to the surface compete for binding of a solution binding ligandwith a directly or indirectly attached recruitment linker. In this case,a loss of signal may be seen.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes. By “target analyte” or“analyte” or grammatical equivalents herein is meant any molecule orcompound to be detected and that can bind to a binding species, definedbelow. Suitable analytes include, but not limited to, small chemicalmolecules such as environmental or clinical chemical or pollutant orbiomolecule, including, but not limited to, pesticides, insecticides,toxins, therapeutic and abused drugs, hormones, antibiotics, antibodies,organic materials, etc. Suitable biomolecules include, but are notlimited to, proteins (including enzymes, immunoglobulins andglycoproteins), nucleic acids, lipids, leggs, carbohydrates, hormones,whole cells (including procaryotic (such as pathogenic bacteria) andeucaryotic cells, including mammalian tumor cells), viruses, spores,etc. Particularly preferred analytes are proteins including enzymes;drugs, cells; antibodies; antigens; cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands.

By “proteins” or grammatical equivalents herein is meant proteins,oligopeptides and peptides, and analogs, including proteins containingnon-naturally occurring amino acids and amino acid analogs, andpeptidomimetic structures.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et W., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nuci. Acids Res.14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Sac. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnonribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribosephosphate backbone may be done to facilitatethe addition of ETMs, or to increase the stability and half-life of suchmolecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogsmay be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. This is particularly advantageous in the systems of thepresent invention, as a reduced salt hybridization solution has a lowerFaradaic current than a physiological salt solution (in the range of 150mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and riba nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

In one embodiment, nucleic acid target analytes are not preferred.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes. In a preferred embodiment,the compositions comprise an electrode comprising a monolayer. By“electrode” herein is meant a composition, which, when connected to anelectronic device, is able to sense a current or charge and convert itto a signal. Thus, an electrode is an ETM as described herein. Preferredelectrodes are known in the art and include, but are not limited to,certain metals and their oxides, including gold; platinum; palladium;silicon; aluminum; metal oxide electrodes including platinum oxide,titanium oxide, tin oxide, indium tin oxide, palladium oxide, siliconoxide, aluminum oxide, molybdenum oxide (Mo206), tungsten oxide (W03)and ruthenium oxides; and carbon (including glassy carbon electrodes,graphite and carbon paste). Preferred electrodes include gold, silicon,carbon and metal oxide electrodes, with gold being particularlypreferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs comprising conductiveoligomers and nucleic acids-bound to the inner surface. This allows amaximum of surface area containing the nucleic acids to be exposed to asmall volume of sample.

The electrode comprises a monolayer, comprising conductive oligomers. By“monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules are oriented approximately parallel toeach other and roughly perpendicular to the surface. Each of themolecules includes a functional group that adheres to the surface, and aportion that interacts with neighboring molecules in the monolayer toform the relatively ordered array. A “mixed” monolayer comprises aheterogeneous monolayer, that is, where at least two different moleculesmake up the monolayer. The SAM may comprise conductive oligomers alone,or a mixture of conductive oligomers and insulators. As outlined herein,the use of a monolayer reduces the amount of non-specific binding ofbiomolecules to the surface, and, in the case of nucleic acids,increases the efficiency of oligonucleotide hybridization as a result ofthe distance of the oligonucleotide from the electrode. Thus, amonolayer facilitates the maintenance of the target analyte away fromthe electrode surface. In addition, a monolayer serves to keep chargecarriers away from the surface of the electrode. Thus, this layer helpsto prevent electrical contact between the electrodes and the ETMs, orbetween the electrode and charged species within the solvent Suchcontact can result in a direct “short circuit” or an indirect shortcircuit via charged species which may be present in the sample.Accordingly, the monolayer is preferably tightly packed in a uniformlayer on the electrode surface, such that a minimum of “holes” exist.The monolayer thus serves as a physical barrier to block solventaccessibility to the electrode.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transferring electronsat 100 Hz. Generally, the conductive oligomer has substantiallyoverlapping n-orbitals, i.e. conjugated n-orbitals, as between themonomeric units of the conductive oligomer, although the conductiveoligomer may also contain one or more sigma (a) bonds. Additionally, aconductive oligomer may be defined functionally by its ability to injector receive electrons into or from an associated ETM. Furthermore, theconductive oligomer is more conductive than the insulators as definedherein. Additionally, the conductive oligomers of the invention are tobe distinguished from electroactive polymers, that themselves may donateor accept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹ cm⁻¹, with from about10⁻⁵ to about 10³ Ω⁻¹ cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 Å to about 200 Å. Asdescribed below, insulators have a conductivity S of about 10⁷ Ω⁻¹ cm⁻¹or lower, with less than about 10⁻⁸ Ω⁻¹ cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during nucleic acidsynthesis (such that nucleosides containing the conductive oligomers maybe added to a nucleic acid synthesizer during the synthesis of thecompositions of the invention), ii) during the attachment of theconductive oligomer to an electrode, or iii) during hybridizationassays. In addition, conductive oligomers that will promote theformation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to capture binding ligands such as nucleic acids, or to several ofthese. Unless otherwise noted, the conductive oligomers depicted hereinwill be attached at the left side to an electrode; that is, as depictedin Structure 1, the left “Y” is connected to the electrode as describedherein. If the conductive oligomer is to be attached to a bindingligand, the right “Y”, if present, is attached to the capture bindingligand, either directly or through the use of a linker, as is describedherein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a conjugated bond, preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyi, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms [C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and ieterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with ieteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- and sulfacompounds, thiols (—SH and —SR), and sulfides (—RSR—). By “phosphoruscontaining moieties” herein is meant compounds containing phosphorus,including, but not limited to, phosphines and phosphates. By “siliconcontaining moieties” herein is meant compounds containing silicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄—CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH. By“amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂), group, although each carbon atom of the ethylene group mayalso be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with R asdescribed above. Ethylene glycol derivatives with other heteroatoms inplace of oxygen (i.e. —(NCH₂—CH₂), or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄—CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated ri-orbitals.

Preferred B-D bonds are selected from acetylene (—C C—, also calledalkyne or ethyne), alkene (CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NHCO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and -0—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably I andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for analyte—binding ligand binding, when the capturebinding ligand is attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient association of binding ligandsand targets, the reaction should occur at a distance from the surface.Thus, for example, for nucleic acid hybridization of target nucleicacids to capture probes on a surface, the hybridization should occur ata distance from the surface, i.e. the kinetics of hybridization increaseas a function of the distance from the surface, particularly for longoligonucleotides of 200 to 300 basepairs. Accordingly, when a nucleicacid is attached via a conductive oligomer, as is more fully describedbelow, the length of the conductive oligomer is such that the closestnucleotide of the nucleic acid is positioned from about 6 Å to about 100Å (although distances of up to 500 Å may be used) from the electrodesurface, with from about 15 Å to about 60 Å being preferred and fromabout 25 Å to about 60 Å also being preferred. Accordingly, n willdepend on the size of the aromatic group, but generally will be fromabout 1 to about 20, with from about 2 to about 15 being preferred andfrom about 3 to about 10 being especially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the capture binding ligand eitherdirectly or via a linker. In some embodiments, for example when theconductive oligomer is attached to a phosphate of the ribose-phosphatebackbone of a nucleic acid, there may be additional atoms, such as alinker, attached between the conductive oligomer and the nucleic acid.Additionally, as outlined below, the D atom may be the nitrogen atom ofthe amino-modified ribose. Alternatively, when m is 1, the conductiveoligomer may terminate in Y, an aromatic group, i.e. the aromatic groupis attached to the capture binding ligand or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF), and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero. Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1. B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isacetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y ispyridine or substituted pyridine and B-D is acetylene; Y is thiophene orsubstituted thiophene and B-D is acetylene; Y is furan or substitutedfuran and B-D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B-D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C-G-C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (-Q=—C—), or alkane (—CR2-CR2-, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be—added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures I and 8.

In addition, the terminus of at least some of the conductive oligomersin the monolayer are electronically exposed. By “electronically exposed”herein is meant that upon the placement of an ETM in close proximity tothe terminus, and after initiation with the appropriate signal, a signaldependent on the presence of the ETM may be detected. The conductiveoligomers may or may not have terminal groups. Thus, in a preferredembodiment, there is no additional terminal group, and the conductiveoligomer terminates with one of the groups depicted in Structures 1 to9; for example, a BD bond such as an acetylene bond. Alternatively, in apreferred embodiment, a terminal group is added, sometimes depictedherein as “Q”. A terminal group may be used for several reasons; forexample, to contribute to the electronic availability of the conductiveoligomer for detection of ETMs, or to alter the surface of the SAM forother reasons, for example to prevent non-specific binding. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when the target analyte is nucleicacid such as DNA or RNA, the nucleic acid is repelled or prevented fromlying down on the surface, to facilitate hybridization. Preferredterminal groups include —NH₂, —OH, —COON, and alkyl groups such as —CH₃,and (poly)alkyloxides such as (poly)ethylene glycol, with —OCH₂CH₂OH,—(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when capture binding ligands are covalently attached to theelectrode using conductive oligomers, it is possible to have one type ofconductive oligomer used to attach the capture binding ligand, andanother type functioning to detect the ETM. Similarly, it may bedesirable to have mixtures of different lengths of conductive oligomersin the monolayer, to help reduce non-specific signals. Thus, forexample, preferred embodiments utilize conductive oligomers thatterminate below the surface of the rest of the monolayer, i.e. below theinsulator layer, if used, or below some fraction of the other conductiveoligomers. Similarly, the use of different conductive oligomers may bedone to facilitate monolayer formation, or to make monolayers withaltered properties.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferably slower than therate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹ cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹ cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that binding is more efficient at adistance from the surface. The species to which capture binding ligandsare attached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the nucleic acidsbeing more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the capture bindingligands are attached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely.Generally, three component systems are preferred, with the first speciescomprising a capture binding ligand containing species (i.e. a captureprobe, that can be attached to the electrode via either an insulator ora conductive oligomer, as is more fully described below). The secondspecies are the conductive oligomers, and the third species areinsulators. In this embodiment, the first species can comprise fromabout 90% to about 1%, with from about 20% to about 40% being preferred.When the capture binding ligands are nucleic acids and the target isnucleic acid as well, from about 30% to about 40% is especiallypreferred for short oligonucleotide targets and from about 10% to about20% is preferred for longer targets. The second species can comprisefrom about 1% to about 90%, with from about 20% to about 90% beingpreferred, and from about 40% to about 60% being especially preferred.The third species can comprise from about 1% to about 90%, with fromabout 20% to about 40% being preferred, and from about 15% to about 30%being especially preferred. Preferred ratios of first: second: thirdspecies are 2:2:1 for short targets, 1:3:1 for longer targets, withtotal thiol concentration in the 500 μM to 1 mM range, and 833 μM beingpreferred.

In a preferred embodiment, two component systems are used, comprisingthe first and second species. In this embodiment, the first species cancomprise from about 90% to about 1%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred.

The covalent attachment of the conductive oligomers and insulators maybe accomplished in a variety of ways, depending on the electrode and thecomposition of the insulators and conductive oligomers used. In apreferred embodiment, the attachment linkers with covalently attachedcapture binding ligands as depicted herein are covalently attached-to anelectrode. Thus, one end or terminus of the attachment linker isattached to the capture binding ligand, and the other is attached to anelectrode. In some embodiments it may be desirable to have theattachment linker attached at a position other than a terminus, or evento have a branched attachment linker that is attached to an electrode atone terminus and to two or more capture binding ligands at othertermini, although this is not preferred. Similarly, the attachmentlinker may be attached at two sites to the electrode, as is generallydepicted in Structures 11-13. Generally, some type of linker is used, asdepicted below as “A” in Structure 10, where “X” is the conductiveoligomer, “I” is an insulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A’ will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313(1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups).

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups.

In a preferred embodiment, the electrode comprising the monolayerincluding conductive oligomers further comprises a capture bindingligand. By “capture binding ligand” or “capture binding species” or“capture probe” herein is meant a compound that is used to probe for thepresence of the target analyte, that will bind to the target analyte.Generally, the capture binding ligand allows the attachment of a targetanalyte to the electrode, for the purposes of detection. As is morefully outlined below, attachment of the target analyte to the captureprobe may be direct (i.e. the target analyte binds to the capturebinding ligand) or indirect (one or more capture extender ligands areused). By “covalently attached” herein is meant that two moieties areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds.

In a preferred embodiment, the binding is specific, and the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding. In someembodiments, for example in the detection of certain biomolecules, thebinding constants of the analyte to the binding ligand will be at leastabout 104-106 M-1, with at least about 105 to 109 M-1 being preferredand at least about 107-109 M-1 being particularly preferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand may be a complementarynucleic acid. Similarly, the analyte may be a nucleic acid bindingprotein and the capture binding ligand is either single-stranded ordouble stranded nucleic acid; alternatively, the binding ligand may be anucleic acid-binding protein when the analyte is a single ordouble-stranded nucleic acid. When the analyte is a protein, the bindingligands include proteins or small molecules. Preferred binding ligandproteins include peptides. For example, when the analyte is an enzyme,suitable binding ligands include substrates and inhibitors. As will beappreciated by those in the art, any two molecules that will associatemay be used, either as an analyte or as the binding ligand. Suitableanalyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligands, proteins/nucleic acid,enzymes/substrates and/or inhibitors, carbohydrates (includingglycoproteins and glycolipids)/lectins, proteins/proteins,proteins/small molecules; and carbohydrates and their binding partnersare also suitable analyte-binding ligand pairs. These may be wild-typeor derivative sequences. In a preferred embodiment, the binding ligandsare portions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor),transferring receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, epidermalgrowth factor receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.

The method of attachment of the capture binding ligand to the attachmentlinker will generally be done as is known in the art, and will depend onthe composition of the attachment linker and the capture binding ligand.In general, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or through theuse of a linker, sometimes depicted herein as “Z”. Linkers are known inthe art; for example, homo-or hetero-bifunctional linkers as are wellknown (see 1994 Pierce Chemical. Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Preferred Z linkers include, but are not limited to, alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred. Z mayalso be a sulfone group, forming sulfonamide.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

In a preferred embodiment, the capture binding ligand is attacheddirectly to the electrode as outlined herein, for example via anattachment linker. Alternatively, the capture binding ligand may utilizea capture extender component, such as depicted in FIG. 2C. In thisembodiment, the capture binding ligand comprises a first portion thatwill bind the target analyte and a second portion that can be used forattachment to the surface. FIG. 2C depicts the use of a nucleic acidcomponent for binding to the surface, although this can be other bindingpartners as well.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand. “Protein” in this context includesproteins, polypeptides and peptides. A wide variety of techniques areknown to add moieties to proteins. One preferred method is outlined inU.S. Pat. No. 5,620,850, hereby incorporated by reference in itsentirety. The attachment of proteins to electrodes is known; see alsoHeller, Acc. Chem. Res. 23:128 (1990), and related work.

A preferred embodiment utilizes nucleic acids as the capture bindingligand, for example for when the target analyte is a nucleic acid or anucleic acid binding protein, or when the nucleic acid serves as anaptamer for binding a protein; see U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, hereby incorporated by reference. In this embodiment, thenucleic acid capture binding ligand is covalently attached to theelectrode, via an “attachment linker”, that can be either a conductiveoligomer or via an insulator. Thus, one end of the attachment linker isattached to a nucleic acid, and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode. Thus, any of structures 1-16 mayfurther comprise a nucleic acid effectively as a terminal group. Thus,the present invention provides compositions comprising binding ligandscovalently attached to electrodes as is generally depicted below inStructure 17 for a nucleic acid:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F, isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the binding ligand, a nucleic acid in Structure 17, andmay be a bond, an atom or a linkage as is herein described. F₂ may bepart of the conductive oligomer, part of the insulator, part of thebinding ligand, or exogeneous to both, for example, as defined hereinfor “Z”.

In general, the methods, synthetic schemes and compositions useful forthe attachment of capture binding ligands, particularly nucleic acids,are outlined in WO98120162, PCT US98/12430, PCT US98/12082; PCTUS99/01705 and PCT US99/01703, all of which are expressly incorporatedherein by reference in their entirety.

In a preferred embodiment, the capture binding ligand is covalentlyattached to the electrode via a conductive oligomer. The covalentattachment of the binding ligand and the conductive oligomer may beaccomplished in several ways, as will be appreciated by those in theart.

In a preferred embodiment, the capture binding ligand is a nucleic acid,and the attachment is via attachment to the base of the nucleoside, viaattachment to the backbone of the nucleic acid (either the ribose, thephosphate, or to an analogous group of a nucleic acid analog backbone),or via a transition metal ligand, as described below. The techniquesoutlined below are generally described for naturally occurring nucleicacids, although as will be appreciated by those in the art, similartechniques may be used with nucleic acid analogs.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the otigomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the base will vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization, it ispreferred to attach at positions not involved in hydrogen bonding to thecomplementary base. Thus, for example, generally attachment is to the 5or 6 position of pyrimidines such as uridine, cytosine and thymine. Forpurines such as adenine and guanine, the linkage is preferably via the 8position. Attachment to non-standard bases is preferably done at thecomparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive otigomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 18 is an example ofthis linkage, using a Structure 3 conductive otigomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide and amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer.

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 10 atoms, with from 1 to 5 atoms being preferred, thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C2 alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Org. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures 3 and 9 conductive oligomers, respectively, using thenitrogen as the heteroatom, although other heteroatoms can be used:

In Structure 21, preferably both m and tare not zero. A preferred Z hereis a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible). Structures 23 and24 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 23 and 24 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B-Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 25. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 26. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure3 conductive oligomers, although other oligomers may be utilized.Structures 25 and 26 depict two representative structures for nucleicacids; as will be appreciated by those in the art, it is possible toconnect other types of capture binding ligands, for exampleproteinaceous binding ligands, in a similar manner:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending-on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (a) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (n)donors, and depicted herein as Lm). Suitable nitrogen donating ligandsare well known in the art and include, but are not limited to, NH2; NHR;NRR′; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine andsubstituted derivatives of bipyridine; terpyridine and substitutedderivatives; phenanthrolines, particularly 1,10-phenanthroline(abbreviated phen) and substituted derivatives of phenanthrolines suchas 4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-cjphenazine(abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene(abbreviated hat); 9,10phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with 6-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith n-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)_(j) and various ring substituted andring fused derivatives, such as the indenylide (−1) ion, that yield aclass of bis (cyclopentadieyl) metal compounds, (i.e. the metallocenes);see for example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982);and Gassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporatedby reference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis (arene)metal compounds and their ring substituted and ringfused derivatives, of which bis (benzene)chromium is a prototypicalexample, Other acyclic n-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjunction with other n-bonded and 5bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 3are depicted in Structures 27 (using phenanthroline and amino asrepresentative ligands), 28 (using ferrocene as the metal-ligandcombination) and 29 (using cyclopentadienyl and amino as representativeligands).

In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

The use of metal ions to connect the binding ligands such as nucleicacids can serve as an internal control or calibration of the system, toevaluate the number of available binding ligands on the surface.However, as will be appreciated by those in the art, if metal ions areused to connect the binding ligands such as nucleic acids to theconductive oligomers, it is generally desirable to have this metal ioncomplex have a different redox potential than that of the ETMs used inthe rest of the system, as described below. This is generally true so asto be able to distinguish the presence of the capture probe from thepresence of the target analyte. This may be useful for identification,calibration and/or quantification. Thus, the amount of capture probe onan electrode maybe compared to the amount of target analyte to quantifythe amount of target sequence in a sample. This is quite significant toserve as an internal control of the sensor or system. This allows ameasurement either prior to the addition of target or after, on the samemolecules that will be used for detection, rather than rely on a similarbut different control system. Thus, the actual molecules that will beused for the detection can be quantified prior to any experiment. Thisis a significant advantage over prior methods.

In a preferred embodiment, the capture binding ligands are covalentlyattached to the electrode via an insulator. The attachment of a varietyof binding ligands such as proteins and nucleic acids to insulators suchas alkyl groups is well known, and can be done to the nucleic acid basesor the backbone, including the ribose or phosphate for backbonescontaining these moieties, or to alternate backbones for nucleic acidanalogs, or to the side chains or backbone of the amino acids.

In a preferred embodiment, there may be one or more different capturebinding ligand species (sometimes referred to herein as “anchorligands”, “anchor probes” or “capture probes” with the phrase “probe”generally referring to nucleic acid species) on the surface, as isgenerally depicted in the Figures. In some embodiments, there may be onetype of capture binding ligand, or one type of capture binding ligandextender, as is more fully described below. Alternatively, differentcapture binding ligands, or one capture binding ligand with amultiplicity of different capture extender binding ligands can be used.Similarly, when nucleic acid systems are used, it may be desirable touse auxiliary capture probes that comprise relatively short probesequences, that can be used to “tack down” components of the system, forexample the recruitment linkers, to increase the concentration of ETMsat the surface.

Thus the present invention provides electrodes comprising monolayerscomprising conductive oligomers and capture binding ligands, useful intarget analyte detection systems.

In a preferred embodiment, the compositions further comprise a solutionbinding ligand. Solution binding ligands are similar to capture bindingligands, in that they bind to target analytes. The solution bindingligand may be the same or different from the capture binding ligand.Generally, the solution binding ligands are not directly attached to thesurface, although as depicted in FIG. 5A they may be. The solutionbinding ligand either directly comprises a recruitment linker thatcomprises at least one ETM, or the recruitment linker is part of a labelprobe that will bind to the solution binding ligand.

Thus, “recruitment linkers” or “signal carriers” with covalentlyattached ETMs are provided. The terms “electron donor moiety”, “electronacceptor moiety”, and “ETMs” (ETMs) or grammatical equivalents hereinrefers to molecules capable of electron transfer under certainconditions. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible electron donor moieties and electronacceptor moieties is very large, and that one skilled in the art ofelectron transfer compounds will be able to utilize a number ofcompounds in the present invention. Preferred ETMs include, but are notlimited to, transition metal complexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventionare listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,N-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def6,5,10-d′e′f′ diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino10-methylphenoxyazine chloride), methyleneblue; Nile blue A (aminoaphthodiethylaminophenoxazine sulfate),indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonic acid;phenosafranine, indigo-5monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and substituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific ETMs will be influenced by the type ofelectron transfer detection used, as is generally outlined below.Preferred ETMs are metallocenes, with ferrocene being particularlypreferred.

In a preferred embodiment, a plurality of ETMs are used. As is shown inthe examples, the use of multiple ETMs provides signal amplification andthus allows more sensitive detection limits. Accordingly, pluralities ofETMs are preferred, with at least about 2 ETMs per recruitment linkerbeing preferred, and at least about 10 being particularly preferred, andat least about 20 to 50 being especially preferred. In some instances,very large numbers of ETMs (100 to 1000) can be used.

As will be appreciated by those in the art, the portion of the labelprobe (or target, in some embodiments) that comprises the ETMs (termedherein a “recruitment linker” or “signal carrier”) can be nucleic acid,or it can be a non-nucleic acid linker that links the solution bindingligand to the ETMs. Thus, as will be appreciated by those in the art,there are a variety of configurations that can be used. In a preferredembodiment, the recruitment linker is nucleic acid (including analogs),and attachment the ETMs can be via (1) a base; (2) the backbone,including the ribose, the phosphate, or comparable structures in nucleicacid analogs; (3) nucleoside replacement, described below; or (4)metallocene polymers, as described below. In a preferred embodiment, therecruitment linker is non-nucleic acid, and can be either a metallocenepolymer or an alkyl-type polymer (including heteroalkyl, as is morefully described below) containing ETM substitution groups. These optionsare generally depicted in FIGS. 14A-14C, 15A-15O and 21A-21E.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers, with the latter being preferred.

In addition, as is described below, when the recruitment linker isnucleic acid, it may be desirable to use secondary label probes, thathave a first portion that will hybridize to a portion of the primarylabel probes and a second portion comprising a recruitment linker as isdefined herein. This is generally depicted in FIGS. 3A-3H and 39R; thisis similar to the use of an amplifier probe, except that both theprimary and the secondary label probes comprise ETMs.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined above for attachment of theconductive oligomer. Attachment can be to an internal nucleoside or aterminal nucleoside.

The covalent attachment to the base will depend in part on the ETMchosen, but in general is similar to the attachment of conductiveoligomers to bases, as outlined above. Attachment may generally be doneto any position of the base. In a preferred embodiment, the ETM is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the ETM. Alternatively,similar types of linkages may be used for the attachment of organicETMs, as will be appreciated by those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp2 and sp Carbon Centers, Sonogashira, pp 521-549, and pp950-953, hereby incorporated by reference). Structure 30 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 30 depicts uridine, although as for all thestructures herein, any other base may also be used.

L, is a ligand, which may include nitrogen, oxygen, sulfur or phosphorusdonating ligands or organometallic ligands such as metallocene ligands.Suitable L, ligands include, but not limited to, phenanthroline,imidazole, bpy and terpy. L, and M are as defined above. Again, it willbe appreciated by those in the art, a linker (“Z”) may be includedbetween the nucleoside and the ETM.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 31, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with L, and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L, of Structure 31 are both metalloceneligands, Lm, as described above. Structure 32 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 32 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z’may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize attachment at the 2′ or 3′ positionof the ribose, with the 2′ position being preferred. Thus for example,the conductive oligomers depicted in Structure 13, 14 and 15 may bereplaced by ETMs; alternatively, the ETMs may be added to the freeterminus of the conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 33. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 34.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 35.

In Structure 35, Z is a linker, as defined herein, and t is either oneor zero. Preferred Z linkers include alkyl groups including heteroalkylgroups such as (CH₂)n and (CH₂CH₂O)n, with n from 1 to 10 beingpreferred, and n=I to 4 being especially preferred, and n=4 beingparticularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in Structures 23 and 24 may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 36.

In Structure 361, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n=1 to 4 being especiallypreferred, and n=4 being particularly preferred.

When the ETM is attached to the base or the backbone of the nucleoside,it is possible to attach the ETMs via “dendrimer” structures, as is morefully outlined below. As is generally depicted in FIG. 2O, alkyl-basedlinkers can be used to create multiple branching structures comprisingone or more ETMs at the terminus of each branch. Generally, this is doneby creating branch points containing multiple hydroxy groups, whichoptionally can then be used to add additional branch points. Theterminal hydroxy groups can then be used in phosphoramidite reactions toadd ETMs, as is generally done below for the nucleoside replacement andmetallocene polymer reactions.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 37(nucleic acid with a ribose-phosphate backbone) and Structure 38(peptide nucleic acid backbone). Structures 37 and 38 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 37, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different Structure 37 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 38, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictsmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction, creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

Thus, by inserting a metallocene such as ferrocene (or other ETM) intothe backbone of a nucleic acid, nucleic acid analogs are made; that is,the invention provides nucleic acids having a backbone comprising atleast one metallocene. This is distinguished from nucleic acids havingmetallocenes attached to the backbone, i.e. via a ribose, a phosphate,etc. That is, two nucleic acids each made up of a traditional nucleicacid or analog (nucleic acids in this case including a singlenucleoside), may be covalently attached to each other via a metallocene.Viewed differently, a metallocene derivative or substituted metalloceneis provided, wherein each of the two aromatic rings of the metallocenehas a nucleic acid substitutent group.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”; that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). For example,as these nucleoside replacements are generally part of probe sequencesto be hybridized with a substantially complementary nucleic acid, forexample a target sequence or another probe sequence, it is possible toadd substitutent groups to the metallocene rings to facilitate hydrogenbonding to the base or bases on the opposite strand. These may be addedto any position on the metallocene rings. Suitable substitutent groupsinclude, but are not limited to, amide groups, amine groups, carboxylicacids, and alcohols, including substituted alcohols. In addition, thesesubstitutent groups can be attached via linkers as well, although ingeneral this is not preferred.

In addition, substituent groups on an ETM, particularly metallocenessuch as ferrocene, may be added to after the redox properties of theETM. Thus, for example, in some embodiments, as is more fully describedbelow, it may be desirable to have different ETMs attached in differentways (i.e. base or ribose attachment), on different probes, or fordifferent purposes (for example, calibration or as an internalstandard). Thus, the addition of substituent groups on the metallocenemay allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 39:

In Structure 39, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein.

Structure 40 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis (or regular protein synthesis) as isknown in the art and depicted within the Figures and Examples:

In Structure 41, the PG protecting group is suitable for use in peptidenucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below.

In a preferred embodiment, the ETMs are attached as polymers, forexample as metallocene polymers, in a “branched” configuration similarto the “branched DNA” embodiments herein and as outlined in U.S. Pat.No. 5,124,246, using modified functionalized nucleotides. The generalidea is as follows. A modified phosphoramidite nucleotide is generatedthat can ultimately contain a free hydroxy group that can be used in theattachment of phosphoramidite ETMs such as metallocenes. This freehydroxy group could be on the base or the backbone, such as the riboseor the phosphate (although as will be appreciated by those in the art,nucleic acid analogs containing other structures can also be used). Themodified nucleotide is incorporated into a nucleic acid, and any hydroxyprotecting groups are removed, thus leaving the free hydroxyl. Upon theaddition of a phosphoramidite ETM such as a metallocene, as describedabove in structures 39 and 40, ETMs, such as metallocene ETMs, areadded. Additional phosphoramidite ETMs such as metallocenes can beadded, to form “ETM polymers”, including “metallocene polymers” asdepicted in FIGS. 21A-21E with ferrocene. In addition, in someembodiments, it is desirable to increase the solubility of the polymersby adding a “capping” group to the terminal ETM in the polymer; forexample a final phosphate group to the metallocene as is generallydepicted in FIG. 21A-21E. Other suitable solubility enhancing “capping”groups will be appreciated by those in the art It should be noted thatthese solubility enhancing groups can be added to the polymers in otherplaces, including to the ligand rings, for example on the metallocenesas discussed herein.

A preferred embodiment of this general idea is outlined in the Figures.In this embodiment, the 2′ position of a ribose of a phosphoramiditenucleotide is first functionalized to contain a protected hydroxy group,in this case via an oxo-linkage, although any number of linkers can beused, as is generally described herein for Z linkers. The protectedmodified nucleotide is then incorporated via standard phosphoramiditechemistry into a growing nucleic acid. The protecting group is removed,and the free hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 41, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers as is generallydepicted in FIGS. 21A-21E. Preferred embodiments also utilizemetallocene polymers from one to about 50 metallocenes in length, withfrom about 5 to about 20 being preferred and from about 5 to about 10being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo-or heterobifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

The attachment of the recruitment linkers to either the solution bindingligand or the first portion of the label probe will depend on thecomposition of the recruitment linker and of the label and/or bindingligand, as will be appreciated by those in the art. When either thelabel probe or the binding ligand is nucleic acid, nucleic acidrecruitment linkers are generally formed during the synthesis of thefirst species, with incorporation of nucleosides containing ETMs asrequired. Alternatively, the first portion of the label probe or thebinding ligand and the recruitment linker may be made separately, andthen attached. When they are both nucleic acid, there may be anoverlapping section of complementarity, forming a section of doublestranded nucleic acid that can then be chemically cross-linked, forexample by using psoralen as is known in the art.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(i.e. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

It is also possible to have ETMs connected to probe sequences, i.e.sequences designed to hybridize to complementary sequences. Thus, ETMsmay be added to non-recruitment linkers as well. For example, there maybe ETMs added to sections of label probes that do hybridize tocomponents of the assay complex, for example the first portion, or tothe target sequence as outlined above and depicted in FIGS. 2A-2D. TheseETMs may be used for electron transfer detection in some embodiments, orthey may not, depending on the location and system. For example, in someembodiments, when for example the target sequence containing randomlyincorporated ETMs is hybridized directly to the capture probe, as isdepicted in FIGS. 2A-2D, there may be ETMs in the portion hybridizing tothe capture probe. If the capture probe is attached to the electrodeusing a conductive oligomer, these ETMs can be used to detect electrontransfer as has been previously described. Alternatively, these ETMs maynot be specifically detected.

Similarly, in some embodiments, when the recruitment linker is nucleicacid, it may be desirable in some instances to have some or all of therecruitment linker be double stranded. In one embodiment, there may be asecond recruitment linker, substantially complementary to the firstrecruitment linker, that can hybridize to the first recruitment linker.In a preferred embodiment, the first recruitment linker comprises thecovalently attached ETMs. In an alternative embodiment, the secondrecruitment linker contains the ETMs, and the first recruitment linkerdoes not, and the ETMs are recruited to the surface by hybridization ofthe second recruitment linker to the first. In yet another embodiment,both the first and second recruitment linkers comprise ETMs. It shouldbe noted, as discussed above, that nucleic acids comprising a largenumber of ETMs may not hybridize as well, i.e. the T_(m) may bedecreased, depending on the site of attachment and the characteristicsof the ETM. Thus, in general, when multiple ETMs are used on hybridizingstrands, generally there are less than about 5, with less than about 3being preferred, or alternatively the ETMs should be spaced sufficientlyfar apart that the intervening nucleotides can sufficiently hybridize toallow good kinetics.

In one embodiment, when nucleic acid targets and/or binding ligandsand/or recruitment linkers are used, non-covalently attached ETMs may beused. In one embodiment, the ETM is a hybridization indicator.Hybridization indicators serve as an ETM that will preferentiallyassociate with double stranded nucleic acid is added, usuallyreversibly, similar to the method of Milian et al., Anal. Chem.65:2317-2323 (1993); Milian et al., Anal. Chem. 662943-2948 (1994), bothof which are hereby expressly incorporated by reference. In thisembodiment, increases in the local concentration of ETMs, due to theassociation of the ETM hybridization indicator with double strandednucleic acid at the surface, can be monitored using the monolayerscomprising the conductive oligomers. Hybridization indicators includeintercalators and minor and/or major groove binding moieties. In apreferred embodiment, intercalators may be used; since intercalationgenerally only occurs in the presence of double stranded nucleic acid,only in the presence of double stranded nucleic acid will the ETMsconcentrate. Intercalating transition metal complex ETMs are known inthe art. Similarly, major or minor groove binding moieties, such asmethylene blue, may also be used in this embodiment.

Similarly, the systems of the invention may utilize non-covalentlyattached ETMs, as is generally described in Napier et al., Bioconj.Chem. 8:906 (1997), hereby expressly incorporated by reference. In thisembodiment, changes in the redox state of certain molecules as a resultof the presence of DNA (i.e. guanine oxidation by ruthenium complexes)can be detected using the SAMs comprising conductive oligomers as well.

Thus, the present invention provides electrodes comprising monolayerscomprising conductive oligomers, generally including capture bindingligands, and either binding ligands or label probes that will bind tothe binding ligands comprising recruitment linkers containing ETMs.

In a preferred embodiment, the compositions of the invention are used todetect target analytes in a sample. In a preferred embodiment, thetarget analyte is a nucleic acid, and thus detection of target sequencesis done. The term “target sequence” or grammatical equivalents hereinmeans a nucleic acid sequence on a single strand of nucleic acid. Thetarget sequence may be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, RNA including mRNA and rRNA, or others. It may be anylength, with the understanding that longer sequences are more specific.As will be appreciated by those in the art, the complementary targetsequence may take many forms. For example, it may be contained within alarger nucleic acid sequence, i.e. all or part of a gene or mRNA, arestriction fragment of a plasmid or genomic DNA, among others. As isoutlined more fully below, probes are made to hybridize to targetsequences to determine the presence or absence of the target sequence ina sample. Generally speaking, this term will be understood by thoseskilled in the art. The target sequence may also be comprised ofdifferent target domains; for example, a first target domain of thesample target sequence may hybridize to a capture probe or a portion ofcapture extender probe, a second target domain may hybridize to aportion of an amplifier probe, a label probe, or a different capture orcapture extender probe, etc. The target domains may be adjacent orseparated. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification occurring as needed,as will be appreciated by those in the art. In a preferred embodiment,when the target analyte is nucleic acid, amplification may be done,including PCR and other amplification techniques as outlined in PCTUS99/01705, incorporated herein by reference in its entirety.

When the target analyte is a nucleic acid, probes of the presentinvention are designed to be complementary to a target sequence (eitherthe target sequence of the sample or to other probe sequences, as isdescribed below), such that hybridization of the target sequence and theprobes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence. Thus,by “substantially complementary” herein is meant that the probes aresufficiently complementary to the target sequences to hybridize undernormal reaction conditions.

Generally, the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length. Thus, in the structures depicted herein,nucleosides may be replaced with nucleic acids.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal., hereby incorporated by reference. The hybridization conditions mayalso vary when a non-ionic backbone, i.e. PNA is used, as is known inthe art. In addition, cross-linking agents may be added after targetbinding to cross-link, i.e. covalently attach, the two strands of thehybridization complex.

As will be appreciated by those in the art, the nucleic acid systems ofthe invention may take on a large number of different configurations, asis generally depicted in the figures. In general, there are three typesof systems that can be used: (1) systems in which the target analyteitself is labeled with ETMs (i.e. the use of a target analyte analog,for non-nucleic acid systems, or, for nucleic acid systems, the targetsequence is labeled; see FIGS. 6A, 6B and 6C); (2) systems in whichlabel probes (or capture binding ligands with recruitment linkers)directly bind (i.e. hybridize for nucleic acids) to the target analytes(see FIGS. 6D-6H for nucleic acid embodiments and FIGS. 2A and 2C fornon-nucleic acid embodiments); and (3) systems in which label probescomprising recruitment linkers are indirectly bound to the targetanalytes, for example through the use of amplifier probes (see FIGS. 61,6J and 6K for nucleic acid embodiments and FIGS. 2B and 2D fornon-nucleic acid embodiments).

In all three of these systems, it is preferred, although not required,that the target analyte be immobilized on the electrode surface. This ispreferably done using capture binding ligands and optionally one or morecapture extender ligands. When only capture binding ligands areutilized, it is necessary to have unique capture binding ligands foreach target analyte; that is, the surface must be customized to containunique capture binding ligands. Alternatively, the use of captureextender ligands, particularly when the capture extender ligands arecapture extender probes (i.e. nucleic acids) may be used, that allow a“universal” surface, i.e. a surface containing a single type of captureprobe that can be used to detect any target sequence.

Capture extender probes or moieties may take on a variety of differentconformations, depending on the identity of the target analyte and ofthe binding ligands. In a preferred embodiment, the target analyte andthe binding ligand are nucleic acids. In this embodiment, the “captureextender” probes are generally depicted in FIG. 6 and have a firstportion that will hybridize to all or part of the capture probe, and asecond portion that will hybridize to a portion of the target sequence.This then allows the generation of customized soluble probes, which aswill be appreciated by those in the art is generally simpler and lesscostly. As shown herein (e.g. FIG. 6H), two capture extender probes maybe used. This has generally been done to stabilize assay complexes (forexample when the target sequence is large, or when large amplifierprobes (particularly branched or dendrimer amplifier probes) are used.

When the capture binding ligand is not a nucleic acid, capture extendercomponents may still be used. In one embodiment, as depicted in FIG. 2C,the capture binding ligand has an associated capture extender of nucleicacid (although as will be appreciated by those in the art, it could bepart of a binding pair as well), that can be used to target to theelectrode surface. Alternatively, an additional capture extendercomponent can be used, to allow a “generic” surface (see FIG. 1).

In a preferred embodiment, the capture binding ligands are added afterthe formation of the SAM ((4) above). This may be done in a variety ofways, as will be appreciated by those in the art. In one embodiment,conductive oligomers with terminal functional groups are made, withpreferred embodiments utilizing activated carboxylates andisothiocyanates, that will react with primary amines that are put ontothe binding ligand, as is generally depicted in FIG. 7 using anactivated carboxylate and nucleic acid, although other capture ligandsmay be attached in this way as well. These two reagents have theadvantage of being stable in aqueous solution, yet react with primaryalkylamines. This allows the spotting of probes (either capture ordetection probes, or both) using known methods (ink jet, spotting, etc.)onto the surface.

In addition, there are a number of non-nucleic acid methods that can beused to immobilize a capture binding ligand on a surface. For example,binding partner pairs can be utilized; i.e. one binding partner isattached to the terminus of the conductive oligomer, and the other tothe end of the binding ligand. This may also be done without using anucleic acid capture probe; that is, one binding partner serves as thecapture probe and the other is attached to either the target sequence ora capture extender probe. That is, either the target sequence comprisesthe binding partner, or a capture extender probe that will hybridize tothe target sequence comprises the binding partner. Suitable bindingpartner pairs include, but are not limited to, hapten pairs such asbiotin/streptavidin; antigens/antibodies; NTAJhistidine tags; etc. Ingeneral, smaller binding partners are preferred.

In a preferred embodiment, when the target sequence itself is modifiedto contain a binding partner, the binding partner is attached via amodified nucleotide that can be enzymatically attached to the targetsequence, for example during a PCR target amplification step.Alternatively, the binding partner should be easily attached to thetarget sequence.

Alternatively, a capture extender probe may be utilized that has anucleic acid portion for hybridization to the target as well as abinding partner (for example, the-capture extender probe may comprise anon-nucleic acid portion such as an alkyl linker that is used to attacha binding partner). In this embodiment, it may be desirable tocross-link the double-stranded nucleic acid of the target and captureextender probe for stability, for example using psoralen as is known inthe art.

In one embodiment, the target is not bound to the electrode surfaceusing capture binding ligands. In this embodiment, what is important, asfor all the assays herein, is that excess label probes be removed priorto detection and that the assay complex (comprising the recruitmentlinker) be in proximity to the surface. As will be appreciated by thosein the art, this may be accomplished in other ways. For example, theassay complex may be present on beads that are added to the electrodecomprising the monolayer. The recruitment linkers comprising the ETMsmay be placed in proximity to the conductive oligomer surface usingtechniques well known in the art, including gravity settling of thebeads on the surface, electrostatic or magnetic interactions betweenbead components and the surface, using binding partner attachment asoutlined above. Alternatively, after the removal of excess reagents suchas excess label probes, the assay complex may be driven down to thesurface, for example by pulsing the system with a voltage sufficient todrive the assay complex to the surface.

However, preferred embodiments utilize assay complexes attached viacapture binding ligands.

For nucleic acid systems, a preferred embodiments utilize the targetsequence itself containing the ETMs. As discussed above, this may bedone using target sequences that have ETMs incorporated at any number ofpositions, as outlined above. Representative examples are depicted inFIGS. 6A, 6B and 6C. In this embodiment, as for the others of thesystem, the 3′-5′ orientation of the probes and targets is chosen to getthe ETM-containing structures (i.e. recruitment linkers or targetsequences) as close to the surface of the monolayer as possible, and inthe correct orientation. This may be done using attachment viainsulators or conductive oligomers as is generally shown in the Figures.In addition, as will be appreciated by those in the art, multiplecapture probes can be utilized, either in a configuration such asdepicted in FIG. 6C, wherein the 5′-3′ orientation of the capture probesis different, or where “loops” of target form when multiples of captureprobes as depicted in FIGS. 6A and 6B are used.

For nucleic acid systems, a preferred embodiments utilize the labelprobes directly hybridizing to the target sequences, as is generallydepicted in FIGS. 6D-61. In these embodiments, the target sequence ispreferably, but not required to be, immobilized on the surface usingcapture probes, including capture extender probes. Label probes are thenused to bring the ETMs into proximity of the surface of the monolayercomprising conductive oligomers. In a preferred embodiment, multiplelabel probes are used; that is, label probes are designed such that theportion that hybridizes to the target sequence (labeled 41 in thefigures) can be different for a number of different label probes, suchthat amplification of the signal occurs, since multiple label probes canbind for every target sequence. Thus, as depicted in the figures, n isan integer of at least one. Depending on the sensitivity desired, helength of the target sequence, the number of ETMs per label probe, etc.,preferred ranges of n are from 1 to 50, with from about 1 to about 20being particularly preferred, and from about 2 to about 5 beingespecially preferred. In addition, if “generic” label probes aredesired, label extender probes can be used as generally described belowfor use with amplifier probes.

As above, generally in this embodiment the configuration of the systemand the label probes (recruitment linkers) are designed to recruit theETMs as close as possible to the monolayer surface.

In a preferred embodiment, the label probes are bound to the targetanalyte indirectly. That is, the present invention finds use in novelcombinations of signal amplification technologies and electron transferdetection on electrodes, which may be particularly useful in sandwichhybridization assays, for nucleic acid detection, as generally depictedin FIG. 61 et seq. In these embodiments, the amplifier probes of theinvention are bound to the target sequence in a sample either directlyor indirectly. Since the amplifier probes preferably contain arelatively large number of amplification sequences that are availablefor binding of label probes, the detectable signal is significantlyincreased, and allows the detection limits of the target to besignificantly improved. These label and amplifier probes, and thedetection methods described herein, may be used in essentially any knownnucleic acid hybridization formats, such as those in which the target isbound directly to a solid phase or in sandwich hybridization assays inwhich the target is bound to one or more nucleic acids that are in turnbound to the solid phase.

In general, these embodiments may be described as follows. An amplifierprobe is hybridized to the target sequence, either directly (e.g. FIG.61), or through the use of a label extender probe (e.g. FIGS. 6N and60), which serves to allow “generic” amplifier probes to be made. Thetarget sequence is preferably, but not required to be, immobilized onthe electrode using capture probes. Preferably, the amplifier probecontains a multiplicity of amplification sequences, although in someembodiments, as described below, the amplifier probe may contain only asingle amplification sequence. The amplifier probe may take on a numberof different forms; either a branched conformation, a dendrimerconformation, or a linear “string” of amplification sequences. Theseamplification sequences are used to form hybridization complexes withlabel probes, and the ETMs can be detected using the electrode.

Accordingly, the present invention provides assay complexes comprisingat least one amplifier probe. By “amplifier probe” or “nucleic acidmultimer” or “amplification multimer” or grammatical equivalents hereinis meant a nucleic acid probe that is used to facilitate signalamplification. Amplifier probes comprise at least a firstsingle-stranded nucleic acid probe sequence, as defined below, and atleast one single-stranded nucleic acid amplification sequence, with amultiplicity of amplification sequences being preferred. In someembodiments, it is possible to use amplifier binding ligands, that arenon-nucleic acid based but that comprise a plurality of binding sitesfor the later association/binding of label ligands comprisingrecruitment linkers. However, amplifier probes are preferred in nucleicacid systems.

Amplifier probes comprise a first probe sequence that is used, eitherdirectly or indirectly, to hybridize to the target sequence. That is,the amplifier probe itself may have a first probe sequence that issubstantially complementary to the target sequence (e.g. FIG. 61), or ithas a first probe sequence that is substantially complementary to aportion of an additional probe, in this case called a label extenderprobe, that has a first portion that is substantially complementary tothe target sequence (e.g. FIG. 6N). In a preferred embodiment, the firstprobe sequence of the amplifier probe is substantially complementary tothe target sequence, as is generally depicted in FIG. 61.

In general, as for all the probes herein, the first probe sequence is ofa length sufficient to give specificity and stability. Thus generally,the probe sequences of the invention that are designed to hybridize toanother nucleic acid (i.e. probe sequences, amplification sequences,portions or domains of larger probes) are at least about 5 nucleosideslong, with at least about 10 being preferred and at least about 15 beingespecially preferred.

In a preferred embodiment, as is depicted in FIG. 8, the amplifierprobes, or any of the other probes of the invention, may form hairpinstem-loop structures in the absence of their target. The length of thestem double-stranded sequence will be selected such that the hairpinstructure is not favored in the presence of target. The use of thesetype of probes, in the systems of the invention or in any nucleic aciddetection systems, can result in a significant decrease in non-specificbinding and thus an increase in the signal to noise ratio.

Generally, these hairpin structures comprise four components. The firstcomponent is a target binding sequence, i.e. a region complementary tothe target (which may be the sample target sequence or another probesequence to which binding is desired), that is about 10 nucleosideslong, with about 15 being preferred. The second component is a loopsequence, that can facilitate the formation of nucleic acid loops.Particularly preferred in this regard are repeats of GTC, which has beenidentified in Fragile X Syndrome as forming turns. (When PNA analogs areused, turns comprising proline residues may be preferred). Generally,from three to five repeats are used, with four to five being preferred.The third component is a self-complementary region, which has a firstportion that is complementary to a portion of the target sequence regionand a second portion that comprises a first portion of the label probebinding sequence. The fourth component is substantially complementary toa label probe (or other probe, as the case may be). The fourth componentfurther comprises a “sticky end”, that is, a portion that does nothybridize to any other portion of the probe, and preferably containsmost, if not all, of the ETMs. The general structure is depicted in FIG.38. As will be appreciated by those in the art, the any or all of theprobes described herein may be configured to form hairpins in theabsence of their targets, including the amplifier, capture, captureextender, label and label extender probes.

In a preferred embodiment, several different amplifier probes are used,each with first probe sequences that will hybridize to a differentportion of the target sequence. That is, there is more than one level ofamplification; the amplifier probe provides an amplification of signaldue to a multiplicity of labeling events, and several differentamplifier probes, each with this multiplicity of labels, for each targetsequence is used. Thus, preferred embodiments utilize at least twodifferent pools of amplifier probes, each pool having a different probesequence for hybridization to different portions of the target sequence;the only real limitation on the number of different amplifier probeswill be the length of the original target sequence. In addition, it isalso possible that the different amplifier probes contain differentamplification sequences, although this is generally not preferred.

In a preferred embodiment, the amplifier probe does not hybridize to thesample target sequence directly, but instead hybridizes to a firstportion of a label extender probe, as is generally depicted in FIG. 39L.This is particularly useful to allow the use of “generic” amplifierprobes, that is, amplifier probes that can be used with a variety ofdifferent targets. This may be desirable since several of the amplifierprobes require special synthesis techniques. Thus, the addition of arelatively short probe as a label extender probe is preferred. Thus, thefirst probe sequence of the amplifier probe is substantiallycomplementary to a first portion or domain of a first label extendersingle-stranded nucleic acid probe. The label extender probe alsocontains a second portion or domain that is substantially complementaryto a portion of the target sequence. Both of these portions arepreferably at least about 10 to about 50 nucleotides in length, with arange of about 15 to about 30 being preferred. The terms “first” and“second” are not meant to confer an orientation of the sequences withrespect to the 5′-3′ orientation of the target or probe sequences. Forexample, assuming a 5′-3′ orientation of the complementary targetsequence, the first portion may be located either 5′ to the secondportion, or 3′ to the second portion. For convenience herein, the orderof probe sequences are generally shown from left to right.

In a preferred embodiment, more than one label extender probe-amplifierprobe pair may be used, that is, n is more than 1. That is, a pluralityof label extender probes may be used, each with a portion that issubstantially complementary to a different portion of the targetsequence; this can serve as another level of amplification. Thus, apreferred embodiment utilizes pools of at least two label extenderprobes, with the upper limit being set by the length of the targetsequence.

In a preferred embodiment, more than one label extender probe is usedwith a single amplifier probe to reduce non-specific binding, as isdepicted in FIG. 60 and generally outlined in U.S. Pat. No. 5,681,697,incorporated by reference herein. In this embodiment, a first portion ofthe first label extender probe hybridizes to a first portion of thetarget sequence, and the second portion of the first label extenderprobe hybridizes to a first probe sequence of the amplifier probe. Afirst portion of the second label extender probe hybridizes to a secondportion of the target sequence, and the second portion of the secondlabel extender probe hybridizes to a second probe sequence of theamplifier probe. These form structures sometimes referred to as“cruciform” structures or configurations, and are generally done toconfer stability when large branched or dendrimeric amplifier probes areused.

In addition, as will be appreciated by those in the art, the labelextender probes may interact with a preamplifier probe, described below,rather than the amplifier probe directly.

Similarly, as outlined above, a preferred embodiment utilizes severaldifferent amplifier probes, each with first probe sequences that willhybridize to a different portion of the label extender probe. Inaddition, as outlined above, it is also possible that the differentamplifier probes contain different amplification sequences, althoughthis is generally not preferred.

In addition to the first probe sequence, the amplifier probe alsocomprises at least one amplification sequence. An “amplificationsequence” or “amplification segment” or grammatical equivalents hereinis meant a sequence that is used, either directly or indirectly, to bindto a first portion of a label probe as is more fully described below.Preferably, the amplifier probe comprises a multiplicity ofamplification sequences, with from about 3 to about 1000 beingpreferred, from about 10 to about 100 being particularly preferred, andabout 50 being especially preferred. In some cases, for example whenlinear amplifier probes are used, from 1 to about 20 is preferred withfrom about 5 to about 10 being particularly preferred. Again, whennon-nucleic acid amplifier moieties are used, the amplification segmentcan bind label ligands.

The amplification sequences may be linked to each other in a variety ofways, as will be appreciated by those in the art. They may be covalentlylinked directly to each other, or to intervening sequences or chemicalmoieties, through nucleic acid linkages such as phosphodiester bonds,PNA bonds, etc., or through interposed linking agents such amino acid,carbohydrate or polyol bridges, or through other cross-linking agents orbinding partners. The site(s) of linkage may be at the ends of asegment, and/or at one or more internal nucleotides in the strand. In apreferred embodiment, the amplification sequences are attached vianucleic acid linkages.

In a-preferred embodiment, branched amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,124,246, hereby incorporated byreference. Branched amplifier probes may take on “fork-like” or“comb-like” conformations. “Fork-like” branched amplifier probesgenerally have three or more oligonucleotide segments emanating from apoint of origin to form a branched structure. The point of origin may beanother nucleotide segment or a multifunctional molecule to which atleast three segments can be covalently or tightly bound. “Comb-like”branched amplifier probes have a linear backbone with a multiplicity ofsidechain oligonucleotides extending from the backbone. In eitherconformation, the pendant segments will normally depend from a modifiednucleotide or other organic moiety having the appropriate functionalgroups for attachment of oligonucleotides. Furthermore, in eitherconformation, a large number of amplification sequences are availablefor binding, either directly or indirectly, to detection probes. Ingeneral, these structures are made as is known in the art, usingmodified multifunctional nucleotides, as is described in U.S. Pat. Nos.5,635,352 and 5,124,246, among others.

In a preferred embodiment, dendrimer amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,175,270, hereby expresslyincorporated by reference. Dendrimeric amplifier probes haveamplification sequences that are attached via hybridization, and thushave portions of double-stranded nucleic acid as a component of theirstructure. The outer surface of the dendrimer amplifier probe has amultiplicity of amplification sequences.

In a preferred embodiment, linear amplifier probes are used, that haveindividual amplification sequences linked end-to-end either directly orwith short intervening sequences to form a polymer. As with the otheramplifier configurations, there may be additional sequences or moietiesbetween the amplification sequences. In addition, as outlined herein,linear amplification probes may form hairpin stem-loop structures, as isdepicted in FIG. 8.

In one embodiment, the linear amplifier probe has a single amplificationsequence. This may be useful when cycles of hybridization/disassociationoccurs, forming a pool of amplifier probe that was hybridized to thetarget and then removed to allow more probes to bind, or when largenumbers of ETMs are used for each label probe. However, in a preferredembodiment, linear amplifier probes comprise a multiplicity ofamplification sequences.

In addition, the amplifier probe may be totally linear, totallybranched, totally dendrimeric, or any combination thereof.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

In addition, the compositions of the invention may include“preamplifier” molecules, which serves a bridging moiety between thelabel extender molecules and the amplifier probes. In this way, moreamplifier and thus more ETMs are ultimately bound to the detectionprobes. Preamplifier molecules may be either linear or branched, andtypically contain in the range of about 30-3000 nucleotides.

The reactions outlined below may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

Generally, the methods are as follows. In a preferred embodiment, thetarget is initially immobilized or attached to the electrode. Fornucleic acids, this is done by forming a hybridization complex between acapture probe and a portion of the target sequence. A preferredembodiment utilizes capture extender probes; in this embodiment, ahybridization complex is formed between a portion of the target sequenceand a first portion of a capture extender probe, and an additionalhybridization complex between a second portion of the capture extenderprobe and a portion of the capture probe. Additional preferredembodiments utilize additional capture probes, thus forming ahybridization complex between a portion of the target sequence and afirst portion of a second capture extender probe, and an additionalhybridization complex between a second portion of the second captureextender probe and a second portion of the capture probe. Non-nucleicacid embodiments utilize capture binding ligands and optional captureextender ligands.

Alternatively, the attachment of the target sequence to the electrode isdone simultaneously with the other reactions.

The method proceeds with the introduction of amplifier probes, ifutilized. In a preferred embodiment, the amplifier probe comprises afirst probe sequence that is substantially complementary to a portion ofthe target sequence, and at least one amplification sequence.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. This will generally be done as is known in the art, anddepends on the type of assay. When the target sequence is immobilized ona surface such as an electrode, the removal of excess reagents generallyis done via one or more washing steps, as will be appreciated by thosein the art. In this embodiment, the target may be immobilized on anysolid support. When the target sequence is not immobilized on a surface,the removal of excess reagents such as the probes of the invention maybe done by adding beads (i.e. solid support particles) that containcomplementary sequences to the probes, such that the excess probes bindto the beads. The beads can then be removed, for example bycentrifugation, filtration, the application of magnetic or electrostaticfields, etc.

The reaction mixture is then subjected to conditions (temperature, highsalt, changes in pH, etc.) under which the amplifier probe disassociatesfrom the target sequence, and the amplifier probe is collected. Theamplifier probe may then be added to an electrode comprising captureprobes for the amplifier probes, label probes added, and detection isachieved.

In a preferred embodiment, a larger pool of probe is generated by addingmore amplifier probe to the target sequence and thehybridization/disassociation reactions are repeated, to generate alarger pool of amplifier probe. This pool of amplifier probe is thenadded to an electrode comprising amplifier capture probes, label probesadded, and detection proceeds.

In this embodiment, it is preferred that the target analyte beimmobilized on a solid support, including an electrode, using themethods described herein; although as will be appreciated by those inthe art, alternate solid support attachment technologies may be used,such as attachment to glass, polymers, etc. It is possible to do thereaction on one solid support and then add the pooled amplifier probe toan electrode for detection.

In a preferred embodiment, the amplifier probe comprises a multiplicityof amplification sequences.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. Again, preferred embodiments utilize immobilized targetsequences, wherein the target sequences are immobilized by hybridizationwith capture probes that are attached to the electrode, or hybridizationto capture extender probes that in turn hybridize with immobilizedcapture probes as is described herein. Generally, in these embodiments,the capture probes and the detection probes are immobilized on theelectrode, generally at the same “address”.

In a preferred embodiment, the first probe sequence of the amplifierprobe is hybridized to a first portion of at least one label extenderprobe, and a second portion of the label extender probe is hybridized toa portion of the target sequence. Other preferred embodiments utilizemore than one label extender probe, as is generally shown in FIG. 60.

In a preferred embodiment, the amplification sequences of the amplifierprobe are used directly for detection, by hybridizing at least one labelprobe sequence.

The invention thus provides assay complexes that minimally comprise atarget sequence and a label probe. “Assay complex” herein is meant thecollection of binding complexes comprising capture binding ligands,target analytes (or analogs, as described below) and label moietiescomprising recruitment linkers that allows detection. The composition ofthe assay complex depends on the use of the different componentsoutlined herein. Thus, in FIG. 6A, the assay complex comprises thecapture probe and the target sequence. The assay complexes may alsoinclude capture extender ligands (including probes), label extenderligands, and amplifier ligands, as outlined herein, depending on theconfiguration used.

The assays are generally run under conditions which allows formation ofthe assay complex only in the presence of target. Stringency can becontrolled by altering a step parameter that is a thermodynamicvariable, including, but not limited to, temperature, formamideconcentration, salt concentration, chaotropic salt concentration pH,organic solvent concentration, etc.

These parameters may also be used to control non-specific binding fornucleic acids, as is generally outlined in U.S. Pat. No. 5,681,697. Thusit may be desirable to perform certain steps at higher stringencyconditions; for example, when an initial hybridization step is donebetween the target sequence and the label extender and capture extenderprobes. Running this step at conditions which favor specific binding canallow the reduction of non-specific binding.

In a preferred embodiment, when all of the components outlined hereinare used, a preferred method for nucleic acid detection is as follows.Single-stranded target sequence is incubated under hybridizationconditions with the capture extender probes and the label extenderprobes. A preferred embodiment does this reaction in the presence of theelectrode with immobilized capture probes, although this may also bedone in two steps, with the initial incubation and the subsequentaddition to the electrode. Excess reagents are washed off, and amplifierprobes are then added. If preamplifier probes are used, they may beadded either prior to the amplifier probes or simultaneously with theamplifier probes. Excess reagents are washed off, and label probes arethen added. Excess reagents are washed off, and detection proceeds asoutlined below.

In one embodiment, a number of capture probes (or capture probes andcapture extender probes) that are each substantially complementary to adifferent portion of the target sequence are used.

Again, as outlined herein, when amplifier probes are used, the system isgenerally configured such that upon label probe binding, the recruitmentlinkers comprising the ETMs are placed in proximity to the monolayersurface. Thus for example, when the ETMs are attached via “dendrimer”type structures as outlined herein, the length of the linkers from thenucleic acid point of attachment to the ETMs may vary, particularly withthe length of the capture probe when capture extender probes are used.That is, longer capture probes, with capture extenders, can result inthe target sequences being “held” further away from the surface than forshorter capture probes. Adding extra linking sequences between the probenucleic acid and the ETMs can result in the ETMs being spatially closerto the surface, giving better results.

In addition, if desirable, nucleic acids utilized in the invention mayalso be ligated together prior to detection, if applicable, by usingstandard molecular biology techniques such as the use of a ligase.Similarly, if desirable for stability, cross-linking agents may be addedto hold the structures stable.

Other embodiments of the invention utilize different steps. For example,competitive assays may be run. In this embodiment, the target analyte ina sample may be replaced by a target analyte analog comprising a portionthat either comprises a recruitment linker or can indirectly bind arecruitment linker. This may be done as is known in the art, for exampleby using affinity chromatography techniques that exchange the analog forthe analyte, leaving the analyte bound and the analog free to interactwith the capture binding ligands on the electrode surface. This isgenerally depicted in FIG. 4A.

Alternatively, a preferred embodiment utilizes a competitive bindingassay when the solution binding ligand comprises a directly orindirectly associated recruitment linker comprising ETMs. In thisembodiment, a target analyte or target analyte analog that will bind thesolution binding ligand is attached to the surface. The solution bindingligand will bind to the surface bound analyte and give a signal. Uponintroduction of the target analyte of the sample, a proportion of thesolution binding ligand will dissociate from the surface bound targetand bind to the incoming target analyte. Thus, a loss of signalproportional to the amount of target analyte in the sample is seen.

The compositions of the invention are generally synthesized as outlinedbelow, generally utilizing techniques well known in the art. As will beappreciated by those in the art, many of the techniques outlined beloware directed to nucleic acids containing a ribose-phosphate backbone.However, as outlined above, many alternate nucleic acid analogs may beutilized, some of which may not contain either ribose or phosphate inthe backbone. In these embodiments, for attachment at positions otherthan the base, attachment is done as will be appreciated by those in theart, depending on the backbone. Thus, for example, attachment can bemade at the carbon atoms of the PNA backbone, as is described below, orat either terminus of the PNA.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to a nucleoside, withaddition of additional nucleosides to form the capture probe followed byattachment to the electrode. Alternatively, the whole capture probe maybe made and then the completed conductive oligomer added, followed byattachment to the electrode. Alternatively, a monolayer of conductiveoligomer (some of which have functional groups for attachment of captureprobes) is attached to the electrode first, followed by attachment ofthe capture probe. The latter two methods may be preferred whenconductive oligomers are used which are not stable in the solvents andunder the conditions used in traditional nucleic acid synthesis.

In a preferred embodiment, the compositions of the invention are made byfirst forming the conductive oligomer covalently attached to thenucleoside, followed by the addition of additional nucleosides to form acapture probe nucleic acid, with the last step comprising the additionof the conductive oligomer to the electrode.

The attachment of the conductive oligomer to the nucleoside may be donein several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode. A number of representative syntheses are shown in theFigures of WO 98/20162, PCT US98/12430, PCT US99/01705 and PCTUS99/01703, all of which are expressly incorporated by reference.

The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone in a number of ways, including attachment viaamide and amine linkages. In a preferred embodiment, there is at least amethylene group or other short aliphatic alkyl groups (as a Z group)between the nitrogen attached to the ribose and the aromatic ring of theconductive oligomer.

Alternatively, attachment is via a phosphate of the ribose-phosphatebackbone.

In a preferred embodiment, attachment is via the base, and can includeacetylene linkages and amide linkages. In a preferred embodiment,protecting groups may be added to the base prior to addition of theconductive oligomers. In addition, the palladium cross-couplingreactions may be altered to prevent dimerization problems; i.e. twoconductive oligomers dimerizing, rather than coupling to the base.

Alternatively, attachment to the base may be done by making thenucleoside with one unit of the oligomer, followed by the addition ofothers.

Once the modified nucleosides are prepared, protected and activated,prior to attachment to the electrode, they may be incorporated into agrowing oligonucleotide by standard synthetic techniques (Gait,Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK1984; Eckstein) in several ways.

In one embodiment, one or more modified nucleosides are converted to thetriphosphate form and incorporated into a growing oligonucleotide chainby using standard molecular biology techniques such as with the use ofthe enzyme DNA polymerise I, T4 DNA polymerase, T7 DNA polymerase, TaqDNA polymerase, reverse transcriptase, and RNA polymerases. For theincorporation of a 3′ modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Thus, the presentinvention provides deoxyribonucleoside triphosphates comprising acovalently attached ETM. Preferred embodiments utilize ETM attachment tothe base or the backbone, such as the ribose (preferably in the 2′position), as is generally depicted below in Structures 42 and 43:

Thus, in some embodiments, it may be possible to generate the nucleicacids comprising ETMs in situ. For example, a target sequence canhybridize to a capture probe (for example on the surface) in such a waythat the terminus of the target sequence is exposed, i.e. unhybridized.The addition of enzyme and triphosphate nucleotides labeled with ETMsallows the in situ creation of the label. Similarly, using labelednucleotides recognized by polymerases can allow simultaneous PCR anddetection; that is, the target sequences are generated in situ.

In a preferred embodiment, the modified nucleoside is converted to thephosphoramidite or Hphosphonate form, which are then used in solid-phaseor solution syntheses of oligonucleotides. In this way the modifiednucleoside, either for attachment at the ribose (i.e. amino- orthiol-modified nucleosides) or the base, is incorporated into theoligonucleotide at either an internal position or the 5′ terminus. Thisis generally done in one of two ways. First, the 5′ position of theribose is protected with 4′,4-dimethoxytrityl (DMT) followed by reactionwith either 2-cyanoethoxy-bisdiisopropylaminophosphine in the presenceof diisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

For attachment of a group to the 3′ terminus, a preferred methodutilizes the attachment of the modified nucleoside (or the nucleosidereplacement) to controlled pore glass (CPG) or other oligomericsupports. In this embodiment, the modified nucleoside is protected atthe Vend with DIVIT, and then reacted with succinic anhydride withactivation. The resulting succinyl compound is attached to CPG or otheroligomeric supports as is known in the art. Further phosphoramiditenucleosides are added, either modified or not, to the Vend afterdeprotection. Thus, the present invention provides conductive oligomersor insulators covalently attached to nucleosides attached to solidoligomeric supports such as CPG, and phosphoramidite derivatives of thenucleosides of the invention.

The invention further provides methods of making label probes withrecruitment linkers comprising ETMs. These synthetic reactions willdepend on the character of the recruitment linker and the method ofattachment of the ETM, as will be appreciated by those in the art. Fornucleic acid recruitment linkers, the label probes are generally made asoutlined herein with the incorporation of ETMs at one or more positions.When a transition metal complex is used as the ETM, synthesis may occurin several ways. In a preferred embodiment, the ligand(s) are added to anucleoside, followed by the transition metal ion, and then thenucleoside with the transition metal complex attached is added to anoligonucleotide, i.e. by addition to the nucleic acid synthesizer.Alternatively, the ligand(s) may be attached, followed by incorporationinto a growing oligonucleotide chain, followed by the addition of themetal ion.

In a preferred embodiment, ETMs are attached to a ribose of theribose-phosphate backbone. This is generally done as is outlined hereinfor conductive oligomers, as described herein, and in PCT publication WO95/15971, using amino-modified or oxo-modified nucleosides, at eitherthe 2′ or 3′ position of the ribose. The amino group may then be usedeither as a ligand, for example as a transition metal ligand forattachment of the metal ion, or as a chemically functional group thatcan be used for attachment of other ligands or organic ETMs, for examplevia amide linkages, as will be appreciated by those in the art. Forexample, the examples describe the synthesis of nucleosides with avariety of ETMs attached via the ribose.

In a preferred embodiment, ETMs are attached to a phosphate of theribose-phosphate backbone. As outlined herein, this may be done usingphosphodiester analogs such as phosphoramidite bonds, see generally PCTpublication WO 95/15971, or the figures.

Attachment to alternate backbones, for example peptide nucleic acids oralternate phosphate linkages will be done as will be appreciated bythose in the art.

In a preferred embodiment, ETMs are attached to a base of thenucleoside. This may be done in a variety of ways. In one embodiment,amino groups of the base, either naturally occurring or added as isdescribed herein (see the figures, for example), are used either asligands for transition metal complexes or as a chemically functionalgroup that can be used to add other ligands, for example via an amidelinkage, or organic ETMs. This is done as will be appreciated by thosein the art Alternatively, nucleosides containing halogen atoms attachedto the heterocyclic ring are commercially available. Acetylene linkedligands may be added using the halogenated bases, as is generally known;see for example, Tzalis et al., Tetrahedron Lett. 36(34):6017-6020(1995); Tzalis et al., Tetrahedron Lett. 36(2):3489-3490 (1995); andTzalis et al., Chem. Communications (in press) 1996, all of which arehereby expressly incorporated by reference. See also the figures and theexamples, which describes the synthesis of metallocenes (in this case,ferrocene) attached via acetylene linkages to the bases.

In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

Once the nucleic acids of the invention are made, with a covalentlyattached attachment linker (i.e. either an insulator or a conductiveoligomer), the attachment linker is attached to the electrode. Themethod will vary depending on the type of electrode used. As isdescribed herein, the attachment linkers are generally made with aterminal “A” linker to facilitate attachment to the electrode. For thepurposes of this application, a sulfur-gold attachment is considered acovalent attachment.

In a preferred embodiment, conductive oligomers, insulators, andattachment linkers are covalently attached via sulfur linkages to theelectrode. However, surprisingly, traditional protecting groups for useof attaching molecules to gold electrodes are generally not ideal foruse in both synthesis of the compositions described herein and inclusionin oligonucleotide synthetic reactions. Accordingly, -the presentinvention provides novel methods for the attachment of conductiveoligomers to gold electrodes, utilizing unusual protecting groups,including ethylpyridine, and trimethylsilylethyl as is depicted in theFigures. However, as will be appreciated by those in the art, when theconductive oligomers do not contain nucleic acids, traditionalprotecting groups such as acetyl groups and others may be used. SeeGreene et al., supra.

This may be done in several ways. In a preferred embodiment, the subunitof the conductive oligomer Which contains the sulfur atom for attachmentto the electrode is protected with an ethyl-pyridine ortrimethylsilylethyl group. For the former, this is generally done bycontacting the subunit containing the sulfur atom (preferably in theform of a sulfhydryl) with a vinyl pyridine group or vinyltrimethylsilylethyl group under conditions whereby an ethylpyridinegroup or trimethylsilylethyl group is added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a nucleoside, and additional nucleosides attached. The protectinggroup is then removed and the sulfur-gold covalent attachment is made.Alternatively, all or part of the conductive oligomer is made, and theneither a subunit containing a protected sulfur atom is added, or asulfur atom is added and then protected. The conductive oligomer is thenattached to a nucleoside, and additional nucleosides attached.Alternatively, the conductive oligomer attached to a nucleic acid ismade, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine or trimethylsilylethyl group mayserve as the protecting group for some of the synthetic reactions, andthen removed and replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure I oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first nucleoside to the conductive oligomer; 4) adding additionalnucleosides to the first nucleoside to form a nucleic acid; 5) attachingthe conductive oligomer to the gold electrode. This may also be done inthe absence of nucleosides.

The above methods may also be used to attach insulator molecules to agold electrode, and moieties comprising capture binding ligands.

In a preferred embodiment, a monolayer comprising conductive oligomers(and preferably insulators) is added to the electrode. Generally, thechemistry of addition is similar to or the same as the addition ofconductive oligomers to the electrode, i.e. using a sulfur atom forattachment to a gold electrode, etc. Compositions comprising monolayersin addition to the conductive oligomers covalently attached to nucleicacids may be made in at least one of five ways: (1) addition of themonolayer, followed by subsequent addition of the attachmentlinker-nucleic acid complex; (2) addition of the attachmentlinker-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and attachment linker-nucleicacid complex; (4) formation of a monolayer (using any of 1, 2 or 3)which includes attachment linkers which terminate in a functional moietysuitable for attachment of a completed nucleic acid; or (5) formation ofa monolayer which includes attachment linkers which terminate in afunctional moiety suitable for nucleic acid synthesis, i.e. the nucleicacid Is synthesized on the surface of the monolayer as is known in theart. Such suitable functional moieties include, but are not limited to,nucleosides, amino groups, carboxyl groups, protected sulfur moieties,or hydroxyl groups for phosphoramidite additions. The examples describethe formation of a monolayer on a gold electrode using the preferredmethod (1).

In a preferred embodiment, the nucleic acid is a peptide nucleic acid oranalog. In this embodiment, the invention provides peptide nucleic acidswith at least one covalently attached ETM or attachment linker. In apreferred embodiment, these moieties are covalently attached to anmonomeric subunit of the PNA. By “monomeric subunit of PNA” herein ismeant the —NH—CH₂—CH₂—N(COCH₂-Base)-CH₂—CO-monomer, or derivatives(herein included within the definition of “nucleoside”) of PNA. Forexample, the number of carbon atoms in the PNA backbone may be altered;see generally Nielsen et al., Chem. Soc. Rev. 1997 page 73, whichdiscloses a number of PNA derivatives, herein expressly incorporated byreference. Similarly, the amide bond linking the base to the backbonemay be altered; phosphoramide and sulfuramide bonds may be used.Alternatively, the moieties are attached to an internal monomericsubunit. By “internal” herein is meant that the monomeric subunit is noteither the N-terminal monomeric subunit or the C-terminal monomericsubunit. In this embodiment, the moieties can be attached either toabase or to the backbone of the monomeric subunit. Attachment to thebase is done as outlined herein or known in the literature. In general,the moieties are added to a base which is then incorporated into a PNAas outlined herein. The base may be either protected, as required forincorporation into the PNA synthetic reaction, or derivatized, to allowincorporation, either prior to the addition of the chemical substituentor afterwards. Protection and derivatization of the bases is shown inthe Figures. The bases can then be incorporated into monomeric subunits;the figures depict two different chemical substituents, an ETM and aconductive oligomer, attached at a base.

In a preferred embodiment, the moieties are covalently attached to thebackbone of the PNA monomer. The attachment is generally to one of theunsubstituted carbon atoms of the monomeric subunit, preferably thea-carbon of the backbone, as is depicted in the Figures, althoughattachment at either of the carbon I or 2 positions, or the a-carbon ofthe amide bond linking the base to the backbone may be done. In the caseof PNA analogs, other carbons or atoms may be substituted as well. In apreferred embodiment, moieties are added at the a-carbon atoms, eitherto a terminal monomeric subunit or an internal one.

In this embodiment, a modified monomeric subunit is synthesized with anETM or an attachment linker, or a functional group for its attachment,and then the base is added and the modified monomer can be incorporatedinto a growing PNA chain. The figures depict the synthesis of aconductive oligomer covalently attached to the backbone of a PNAmonomeric subunit, and the synthesis of a ferrocene attached to thebackbone of a monomeric subunit.

Once generated, the monomeric subunits with covalently attached moietiesare incorporated into a PNA using the techniques outlined in Will etal., Tetrahedron 51(44):12069-12082.(1995), and Vanderlaan et al., Tett.Let. 38:2249-2252 (1997), both of which are hereby expresslyincorporated in their entirety. These procedures allow the addition ofchemical substituents to peptide nucleic acids without destroying thechemical substituents.

As will be appreciated by those in the art, electrodes may be made thathave any combination of nucleic acids, conductive oligomers andinsulators.

The compositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitate cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporated byreference.

Once made, the compositions find use in a number of applications, asdescribed herein. In particular, the compositions of the invention finduse in target analyte assays. As will be appreciated by those in theart, electrodes can be made that have a single species of bindingligands such as nucleic acid, i.e. a single binding ligand, or multiplebinding ligand species.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these probes in an array form. The use ofoligonucleotide arrays are well known in the art, and the methods andcompositions herein allow the use of array formats for other targetanalytes as well. In addition, techniques are known for “addressing”locations within an electrode and for the surface modification ofelectrodes. Thus, in a preferred embodiment, arrays of different bindingligands are laid down on the electrode, each of which are covalentlyattached to the electrode via a conductive linker. In this embodiment,the number of different species may vary widely, from one to thousands,with from about 4 to about 100,000 being preferred, and from about 10 toabout 10,000 being particularly preferred.

The invention finds use in the screening of candidate bioactive agentsfor therapeutic agents that can alter the binding of the analyte to thebinding ligand, and thus may be involved in biological function. Theterm “agent” or “exogenous compound” as used herein describes anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., with the capability of directly orindirectly altering target analyte binding. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds In theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Candidate agents may be added either before or after the target analyte.

Once the assay complexes of the invention are made, that minimallycomprise a target sequence and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem. 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the ETM of the label probe. Thus, at voltages above theredox potential of the input electron source, both the ETM and the inputelectron source are oxidized and can thus donate electrons; the ETMdonates an electron to the electrode and the input source donates to theETM. For example, ferrocene, as a ETM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the ETMattached to the nucleic acid. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the ETM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the ETM are used. At voltages less than the redox potential of theETM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the ETM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the ETM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the ETM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the ETM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the ETM of the label probe.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the ETMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the ETM on the labelprobe. When the ETM is not present, i.e. when the target sequence is nothybridized to the composition of the invention, luminol is notsignificantly oxidized, resulting in a low photon emission and thus alow (if any) signal from the luminol. In the presence of the target, amuch larger signal is generated. Thus, the measure of luminol oxidationby photon emission is an indirect measurement of the ability of the ETMto donate electrons to the electrode. Furthermore, since photondetection is generally more sensitive than electronic detection, thesensitivity of the system may be increased. Initial results suggest thatluminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectral changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluorescence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 ppl 97-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl2-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the FluorImagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277.1988).

Preferred examples display efficient fluorescence (reasonably highquantum yields) as well as low reorganization energies. These includeRu(4,7-biphenyl2-phenanthroline)₃ ²⁺, Ru(4,4′-diphenyl-2,2′bipyridine)₃²⁺ and platinum complexes (see Cummings et al., J. Am. Chem. Soc.118:1949-1960 (1996), incorporated by reference). Alternatively, areduction in fluorescence associated with hybridization can be measuredusing these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺(bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see Blackburn,G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (1)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

Accordingly, alternate equations were developed, using the Nernstequation and first principles to develop a model which more closelysimulates the results. This was derived as follows. The Nernst equation,Equation 1 below, describes the ratio of oxidized (O) to reduced (R)molecules (number of molecules=n) at any given voltage and temperature,since not every molecule gets oxidized at the same oxidation potential.

$\begin{matrix}{{{Equation}\mspace{20mu} 1}{E_{DC} = {E_{0} + {\frac{RT}{nF}\ln\frac{\lbrack O\rbrack}{\lbrack R\rbrack}(0)}}}} & (1)\end{matrix}$

E_(DC) is the electrode potential, E_(o) is the formal potential of themetal complex, R is the gas constant, T is the temperature in degreesKelvin, n is the number of electrons transferred, F is faraday'sconstant, [0] is the concentration of oxidized molecules and [R] is theconcentration of reduced molecules.

The Nemst equation can be rearranged as shown in Equations 2 and 3:

$\begin{matrix}{{E_{DC} - E_{0}} = {\frac{RT}{nF}\ln\frac{\lbrack O\rbrack}{\lbrack R\rbrack}(0)}} & {{Equation}\mspace{20mu} 2}\end{matrix}$

E_(DC) is the DC component of the potential.

$\begin{matrix}{{\exp\;\frac{nF}{RT}\left( {E_{DC} - E_{0}} \right)} = {\frac{\lbrack O\rbrack}{\lbrack R\rbrack}(0)}} & {{Equation}\mspace{20mu} 3}\end{matrix}$

Equation 3 can be rearranged as follows, using normalization of theconcentration to equal 1 for simplicity, as shown in Equations 4, 5 and6. This requires the subsequent multiplication by the total number ofmolecules.[O]+[R]=1  Equation 4[O]=1=[R]  Equation 5[R]=1=[O]  Equation 6

Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RTequals 38.9 V⁻¹, for n=1, gives Equations 7 and 8, which define [0] and[R], respectively:

$\begin{matrix}{\lbrack O\rbrack = {\frac{\exp^{38.9{({E - E_{0}})}}}{1 + \exp^{38.9{({E - E_{0}})}}}(0)}} & {{Equation}\mspace{20mu} 7} \\{\lbrack R\rbrack = {\frac{1}{1 + \exp^{38.9{({E - E_{0}})}}}(0)}} & {{Equation}\mspace{20mu} 8}\end{matrix}$

Taking into consideration the generation of an AC faradaic current, theratio of [O]/[R] at any given potential must be evaluated. At aparticular E_(DC) with an applied E_(AC), as is generally describedherein, at the apex of the E_(AC) more molecules will be in the oxidizedstate, since the voltage on the surface is now (E_(DC)+E_(AC)); at thebottom, more will be reduced since the voltage is lower. Therefore, theAC current at a given E_(DC) will be dictated by both the AC and DCvoltages, as well as the shape of the Nernstian curve. Specifically, ifthe number of oxidized molecules at the bottom of the AC cycle issubtracted from the amount at the top of the AC cycle, the total changein a given AC cycle is obtained, as is generally described by Equation9. Dividing by 2 then gives the AC amplitude.

$\begin{matrix}{i_{AC} \cong {\left( {{electrons}\mspace{14mu}{{at}\mspace{11mu}\left\lbrack {\underset{2}{E_{DC}} + E_{AC}} \right\rbrack}} \right) - \left( {{electrons}\mspace{14mu}{{at}\mspace{14mu}\left\lbrack {E_{DC} - E_{AC}} \right\rbrack}} \right)}} & {{Equation}\mspace{20mu} 9}\end{matrix}$

Equation 10 thus describes the AC current which should result:i _(AC) =C ₀ Fω% ₀([O]_(E) _(DC) _(+E) _(AC) −[O]_(E) _(DC) _(−E) _(AC))(0)  Equation 10

As depicted in Equation 11, the total AC current will be the number ofredox molecules C), times faraday's constant (F), times the AC frequency(ω), times 0.5 (to take into account the AC amplitude), times the ratiosderived above in Equation 7. The AC voltage is approximated by theaverage, E_(AC)2/π.

$\begin{matrix}{i_{AC} = {{\frac{C_{0}F\;\omega}{2}\left( \frac{\exp^{38.9\lbrack{E_{DC} + \frac{2_{E_{AC}}}{\pi} - E_{0}}\rbrack}}{1 + \exp^{38.9\lbrack{E_{DC} + \frac{2_{E_{AC}}}{\pi} - E_{0}}\rbrack}} \right)} - \frac{\exp^{38.9\lbrack{E_{DC} + \frac{2_{E_{AC}}}{\pi} - E_{0}}\rbrack}}{1 + \exp^{38.9\lbrack{E_{DC} + \frac{2_{E_{AC}}}{\pi} - E_{0}}\rbrack}}}} & {{Equation}\mspace{20mu} 11}\end{matrix}$

Using Equation 11, simulations were generated using increasingoverpotential (AC voltage). FIG. 22A shows one of these simulations,while FIG. 22B depicts a simulation based on traditional theory. FIGS.23A and 23B depicts actual experimental data using the Fc-wire ofExample 7 plotted with the simulation, and shows that the model fits theexperimental data very well. In some cases the current is smaller thanpredicted, however this has been shown to be caused by ferrocenedegradation which may be remedied in a number of ways. However, Equation11 does not incorporate the effect of electron transfer rate nor ofinstrument factors. Electron transfer rate is important when the rate isclose to or lower than the applied frequency. Thus, the true i_(AC)should be a function of all three, as depicted in Equation 12.i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 12

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound label probes/ETMs show differences inimpedance (i.e. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined; either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single input highfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of ‘holes’ where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance.

In a preferred embodiment, the output signal comprises an AC current Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa.

Thus, the assay complexes comprising the nucleic acids in this systemhave a certain faradaic impedance, that will depend on the distancebetween the ETM and the electrode, their electronic properties, and thecomposition of the intervening medium, among other things. Of importancein the methods of the invention is that the faradaic impedance betweenthe ETM and the electrode is significantly different depending onwhether the label probes containing the ETMs are specifically ornon-specifically bound to the electrode.

Accordingly, the present invention further provides apparatus for thedetection of nucleic acids using AC detection methods. The apparatusincludes a test chamber which has at least a first measuring or sampleelectrode, and a second measuring or counter electrode. Three electrodesystems are also useful. The first and second measuring electrodes arein contact with a test sample receiving region, such that in thepresence of a liquid test sample, the two electrodes may be inelectrical contact.

In a preferred embodiment, the first measuring electrode comprises asingle stranded nucleic acid capture probe covalently attached via anattachment linker, and a monolayer comprising conductive oligomers, suchas are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber, that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofantiHIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for-extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labeled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labeled with an ETM. Elongation results indouble stranded nucleic acid with a ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding-probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 39. By utilizingcomplementary portions of nucleic acid, while leaving “sticky ends”, anarray comprising any number of restriction endonuclease sites is made.Treating a target sample with one or more of these restrictionendonucleases allows the targets to bind to the array. This can be donewithout knowing the sequence of the target The target sequences can beligated, as desired, using standard methods such as ligases, and thetarget sequence detected, using either standard labels or the methods ofthe invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁵ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transfermoeity) is used for each target sequence, the sensitivity will go up.

While the limits of detection are currently being evaluated, based onthe published electron transfer rate through DNA, which is roughly 1×10⁶electrons/sec/duplex for an 8 base pair separation (see Meade et al.,Angw. Chem. Eng. Ed., 34:352 (1995)) and high driving forces, ACfrequencies of about 100 kHz should be possible. As the preliminaryresults show, electron transfer through these systems is quiteefficient, resulting in nearly 100×10³ electrons/sec, resulting inpotential femptoamp sensitivity for very few molecules.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example I Synthesis of Nucleoside Modified with Ferrocene atthe 2′ Position

The preparation of N6 is described as shown in FIG. 9.

Compound N1. Ferrocene (20 g, 108 mmol) and 4-bromobutyl chloride (20 g,108 mmol) were dissolved in 450 mL dichloromethane followed by theaddition of AICI₃ anhydrous (14.7 g, 11 μmol). The reaction mixture wasstirred at room temperature for 1 hour and 40 minutes, then was quenchedby addition of 600 mL ice. The organic layer was separated and washedwith water until the aqueous layer was close to neutral (pH=5). Theorganic layer was dried with Na₂SO₄ and concentrated. The crude productwas purified by flash chromatography eluting with 50/50hexane/dichloromethane and later 30/70 hexane/dichloromethane on 300 gsilica gel to afford 26.4 gm (73%) of the title product.

Compound N2. Compound N1 (6 g, 18 mmol) was dissolved in 120 mL toluenein a round bottom flask. zinc (35.9 g, 55 mmol), mercuric chloride (3.3g, 12 mmol) and water (100 mL) were added successively. Then HClsolution (12 M, 80 mL) was added dropwise. The reaction mixture wasstirred at room temperature for 16 hours. The organic layer wasseparated, and washed with water (2×100 mL) and concentrated. Furtherpurification by flash chromatography (hexane) on 270 gm of silica gelprovided the desired product as a brown solid (3.3 g, 58%).

Compound N3. A mixture of 13.6 gm (51 mmol) of adenosine in 400 mL dryDMF was cooled in a ice-water bath for 10 minutes before the addition of3.0 gm (76 mmol) of NaH (60%). The reaction mixture was stirred at 0° C.for one hour before addition of Compound N2 (16.4 g, 51 mmol). Then thetemperature was slowly raised to 30° C., and the reaction mixture waskept at this temperature for 4 hours before being quenched by 100 mLice. The solvents were removed in vacuo. The resultant gum was dissolvedin 300 mL water and 300 mL ethyl acetate. The aqueous layer wasextracted thoroughly (3×300 mL ethyl acetate). The combined organicextracts were concentrated, and the crude product was purified by flashchromatography on 270 g silica gel. The column was eluted with 20% ethylacetate/dichloromethane, 50% ethyl acetate/dichloromethane, 70% ethylacetate/dichloromethane, ethyl acetate, 1% methano Vethyl acetate, 3%methano Vethyl acetate, and 5% methanol/ethyl acetate. The concentrationof the desired fractions provide the final product (6.5 g, 25%).

Compound N4. Compound N3 (6.5 g, 12.8 mmol) was dissolved in 150 mL drypyridine, followed by adding TMSCI (5.6 g, 51.2 mmol). The reactionmixture was stirred at room temperature for 1.5 hours. Thenphenoxyacetyl chloride (3.3 g, 19.2 mmol) was added at 0° C. Thereaction was then stirred at room temperature for 4 hours and wasquenched by the addition of 100 mL water at 0° C. The solvents wereremoved under reduced pressure, and the crude gum was further purifiedby flash chromatography on 90 g of silica gel (1%methanol/dichloromethane) (2.3 g, 28%).

Compound N5. Compound N4 (2.2 g, 3.4 mmol) and DMAP (200 mg, 1.6 mmol)were dissolved in 150 mL dry pyridine, followed by the addition of DMTCI(1.4 g, 4.1 mmol). The reaction was stirred under argon at roomtemperature overnight The solvent was removed under reduced pressure,and the residue was dissolved in 250 mL dichloromethane. The organicsolution washed by 5% NaHCO₃ solution (3×250 mL), dried over Na₂SO₄, andconcentrated. Further purification by flash chromatography on 55 g ofsilica gel (1% TEA/50% hexane/dichloromethane) provided the desiredproduct (1.3 g, 41%).

Compound N6. To a solution of N5 (3.30 gm, 3.50 mmol) in 150 mLdichloromethane. Diisopropylethylamine (4.87 mL, 8.0 eq.) and catalyticamount of DMAP (200 mg) were added. The mixture was kept at 0° C., andN,N-diisopropylamino cyanoethyl phosphonamidic chloride (2.34 mL, 10.48mmol) was added. The reaction mixture was warmed up and stirred at roomtemperature overnight. After dilution by adding 150 mL ofdichloromethane and 250 mL of 5% NaHCO₃ aqueous solution, the organiclayer was separated, washed with 5% NaHCO₃ (250 mL), dried over Na₂SO₄,and concentrated. The crude product was purified on a flash column of 66g of silica gel packed with 1% TEA in hexane. The eluting solvents were1% TEA in hexane (500 mL), 1% TEA and 10% dichloromethane in hexane (500mL), 1% TEA and 20% dichloromethane in hexane (500 mL). 1% TEA and 50%dichloromethane in hexane (500 mL). Fractions containing the desiredproducts were collected and concentrated to afford the final product (3gm, 75%).

Example 2 Synthesis of “Branched” Nucleoside

The synthesis of N17 is described as shown in FIG. 10.

Synthesis of N14. To a solution of tert-butyldimethylsily chloride(33.38 g, 0.22 mol) in 300 mL of dichloromethane was added imidazole(37.69 g, 0.55 mol). Immediately, large amount of precipitate wasformed. 2-Bromoethanol (27.68 g, 0.22 mol,.) was added slowly at roomtemperature. The reaction mixture was stirred at this temperature for 3hours. The organic layer washed with water (200 mL), 5% NaHCO₃ (2×250mL), and water (200 mL). The removal of solvent afforded 52.52 g of thetitle product (99%).

Synthesis of N15. To a suspension of adenosine (40 g, 0.15 mol) in 1.0 Lof DMF at 0° C., was added NaH (8.98 gm of 60% in mineral oil, 0.22mol). The mixture was stirred at 0° C. for 1 hour, and N14 (35.79 gm,0.15 mol) was added. The reaction was stirred at 30° C. overnight. Itwas quenched by 100 mL ice-water. The solvents were removed under highvacuum. The resultant foam was dissolved in a mixture of 800 mL of ethylacetate and 700 mL of water. The aqueous layer was further extracted byethyl acetate (3×200 mL). The combined organic layer was dried overNa₂SO₄ and concentrated. The crude product was further purified on aflash column of 300 g of silica gel packed with 1% TEA indichloromethane. The eluting solvents were dichloromethane (500 mL), 3%MeOH in dichloromethane (500 mL), 5% MeOH in dichloromethane (500 mL),and 8% MeOH in dichloromethane (2000 mL). The desired fractions werecollected and concentrated to afford 11.70 g of the title product (19%).

Synthesis of N16. To a solution of N15 (11.50 gm, 27.17 mmol) in 300 mLdry pyridine cooled at 0° C., was added trimethylsily chloride (13.71mL, 0.11 mol, 4.0). The mixture was stirred at 0° C. for 40 min.Phenoxyacetyl chloride (9.38 mL, 67.93 mmol) was added. The reaction wasstirred at 0° C. for 2.5 h. The mixture was then transferred to amixture of 700 mL of dichloromethane and 500 mL water. The mixture wasshaken well and organic layer was separated. After washing twice with 5%NaHCO₃ (2×300 mL), dichloromethane was removed on a rotovapor. Into theresidue was added 200 mL of water, the resulting pyridine mixture wasstirred at room temperature for 2 hours. The solvents were then removedunder high vacuum. The gum product was co-evaporated with 100 mL ofpyridine. The residue was dissolved in 250 mL of dry pyridine at 0° C.,and 4,4′-dimethoxytrityl chloride (11.02 gm, 32.60 mmol) was added. Thereaction was stirred at room temperature overnight. The solution wastransferred to a mixture of 700 mL of dichloromethane and 500 mL of 5%NaHCO₃. After shaking well, the organic layer was separated, furtherwashed with 5% NaHCO₃ (2×200 mL), and then concentrated. The crudeproduct was purified on a flash column of 270 gm of silica gel packedwith 1% TEA/30% CH₂Cl₂/Hexane. The eluting solvents were 1% TEA/50%CH₂Cl₂/Hexane (1000 mL), and 1% TEA/CH₂Cl₂ (2000 mL). The fractionscontaining the desired product were collected and concentrated to afford10.0 g of the title product (43%).

Synthesis of N17. To a solution of N16 (10.0 gm, 11.60 mmol) in 300 mLdichloromethane. Diisopropylethylamine (16.2 mL) and catalytic amount ofN,N-dimethylaminopyridine (200 mg) were added. The mixture was cooled inan ice-water bath, and N,N-diisopropylamino cyanoethyl phosphonamidicchloride (7.78 mL, 34.82 mmol) was added. The reaction was stirred atroom temperature overnight. The reaction mixture was diluted by adding250 mL of dichloromethane and 250 mL of 5% NaHCO₃. After shaking well,the organic layer was separated and washed once more with the sameamount of 5% NaHCO₃ aqueous solution, dried over Na₂SO₄, andconcentrated. The crude product was purified on a flash column of 120 gmof silica gel packed with 1% TEA and 10% dichloromethane in hexane. Theeluting solvents were 1% TEA and 10% dichloromethane in hexane (500 mL),1% TEA and 20% dichloromethane in hexane (500 mL), and 1% TEA and 40%dichloromethane in hexane (1500 mL). The right fractions were collectedand concentrated to afford the final product (7.37 gm, 60%).

Example 3 Synthesis of Nucleoside with Ferrocene Attached Via aPhosphate

The synthesis of Y63 is described as shown in FIG. 11.

Synthesis of C102: A reaction mixture consisting of 10.5 gm (32.7 mmol)of N2, 16 gm of potassium acetate and 350 ml of DMF was stirred at 100°C. for 2.5 hrs. The reaction mixture was allowed to cool to roomtemperature and then poured into a mixture of 400 ml of ether and 800 mlof water. The mixture was shaken and the organic layer was separated.The aqueous layer was extracted twice with ether. The combined etherextracts were dried over sodium sulfate and then concentrated for columnchromatography: Silica gel (160 gm) was packed with 1% TEAIHexane. Thecrude was loaded and the column was eluted with 1% TEA/0-100%CH₂Cl₂/Hexane. Fractions containing desired product were collected andconcentrated to afford 5.8 g (59.1%) of C102.

Synthesis of Y61: To a flask containing 5.1 gm (17.0 mmol) of C102 wasadded 30 ml of Dioxane. To this solution, small aliquots of 1 M NaOH wasadded over a period of 2.5 hours or until hydrolysis was complete. Afterhydrolysis the product was extracted using hexane. The combined extractswere dried over sodium sulfate and concentrated for chromatography.Silica gel (100 gm) was packed in 10% EtOAc/Hexane. The crude productsolution was loaded and the column was eluted with 10% to 50% EtOAc inhexane. The fractions containing desired product were pooled andconcentrated to afford 4.20 gm (96.1%) of Y61.

Synthesis of Y62: To a flask containing 4.10 gm (15.9 mmol) of Y61 wasadded 200 ml of dichloromethane and 7.72 ml of DIPEA and 4.24 gm (15.9mmol) of bis(diisopropylamino) chlorophosphine. This reaction mixturewas stirred under the presence of argon overnight. After the reactionmixture was concentrated to 1/3 of its original volume, 200 ml of hexanewas added and then the reaction mixture was again concentrated to 1/3 isoriginal volume. This procedure was repeated once more. The precipitatedsalts were filtered off and the solution was concentrated to afford 8.24gm of crude Y62. Without further purification, the product was used fornext step.

Synthesis of Y63: A reaction mixture of 1.0 gm (1.45 mmol) of N-PACdeoxy-adenosine, 1.77 g of the crude Y62, and 125 mg ofN,N-diisopropylammonium tetrazolide, and 100 ml of dichloromethane. Thereaction mixture was stirred at room temperature overnight. The reactionmixture was then diluted by adding 100 ml of CH₂Cl₂ and 100 ml of 5%NaHCO₃ solution. The organic phase was separated and dried over sodiumsulfate. The solution was then concentrated for column chromatography.Silica gel (35 gm) was packed with 1% TEA/Hexane. The crude material waseluted with 1% TEA 110-40% CH₂Cl₂/Hexane. The fractions containingproduct were pooled and concentrated to afford 0.25 gm of the titleproduct.

Example 4 Synthesis of Ethylene Glycol Terminated Wire W71

As shown in FIG. 12.

Synthesis of W55: To a flask was added 7.5 gm (27.3 mmol) oftert-butyldiphenylchlorosilane, 25.0 gm (166.5 mmol) of tri(ethyleneglycol) and 50 ml of dry DMF under argon. The mixture was stirred andcooled in an ice-water bath. To the flask was added dropwise a clearsolution of 5.1 gm (30.0 mmol) of AgNO₃ in 80 mL of DMF through anadditional funnel. After the completeness of addition, the mixture wasallowed to warm up to room temperature and was stirred for additional 30min. Brown AgCI precipitate was filtered out and washed with DMF(3×10mL). The removal of solvent under reduced pressure resulted in formationof thick syrup-like liquid product that was dissolved in about 80 ml ofCH₂Cl₂. The solution washed with water (6×100 mL) in order to removeunreacted starting material, i.e., tris (ethylene glycol), then driedover Na₂SO4. Removal of CH₂Cl₂ afforded ˜10.5 g crude product, which waspurified on a column containing 104 g of silica gel packed with 50CH2Cl2/hexane. The column was eluted with 3-5% MeOH/CH₂Cl₂. Thefractions containing the desired product were pooled and concentrated toafford 8.01 gm (75.5%) of the pure title product.

Synthesis of W68: To a flask containing 8.01 gm (20.6.0 mmol) of W55 wasadded 8.56 gm (25.8 mmol) of CBr₄ and 60 ml of CH₂Cl₂. The mixture wasstirred in an ice-water bath. To the solution was slowly added 8.11 gm(31.0 mmol) of PPh₃ in 15 ml CH₂Cl₂. The mixture was stirred for about35 min. at 0° C., and allowed to warm to room temperature. The volume ofthe mixture was reduced to about 10.0 ml and 75 ml of ether was added.The precipitate was filtered out and washed with 2×75 of ether. Removalof ether gave about 15 gm of crude product that was used forpurification. Silica gel (105 gm) was packed with hexane. Upon loadingthe sample solution, the column was eluted with 50% CH₂Cl₂/hexane andthen CH₂Cl₂. The desired fractions were pooled and concentrated to give8.56 gm (72.0%) of pure title product.

Synthesis of W69: A solution of 5.2 gm (23.6 mmol)of 4-iodophenol in 50ml of dry DMF was cooled in an ice-water bath under Ar. To the mixturewas added 1.0 gm of NaH (60% in mineral oil, 25.0 mmol) portion byportion. The mixture was stirred at the same temperature for about 35min. and at room temperature for 30 min. A solution of 8.68 gm (19.2mmol) of W68 in 20 ml of DMF was added to the flask under argon. Themixture was stirred at 50° C. for 12 hr with the flask covered withaluminum foil. DMF was removed under reduced pressure. The residue wasdissolved in 300 ml of ethyl acetate, and the solution washed with H₂O(6×50 mL). Ethyl acetate was removed under reduced pressure and theresidue was loaded into a 100 g silica gel column packed with 30%CH₂Cl₂/hexane for the purification. The column was eluted with 30-100%CH₂Cl₂/hexane. The fractions containing the desired product were pooledand concentrated to afford 9.5 gm (84.0%) of the title product.

Synthesis of W70: To a 100 ml round bottom flask containing 6.89 gm(11.6 mmol) of W69 was added 30 ml of 1 M TBAF THF solution. Thesolution was stirred at room temperature for 5 h. THF was removed andthe residue was dissolved 150 ml of CH₂Cl₂. The solution washed with H₂O(4×25 mL). Removal of solvent gave 10.5 gm of semi-solid. Silica gel (65gm) was packed with 50% CH₂Cl₂/hexane, upon loading the sample solution,the column was eluted with 0-3% CH₃OH/CH₂Cl₂. The fractions wereidentified by TLC(CH₃OH:CH₂Cl₂=5:95). The fractions containing thedesired product were collected and concentrated to afford 4.10 gm(99.0%) of the title product.

Synthesis of W71: To a flask was added 1.12 gm (3.18 mmol) of W70, 0.23g (0.88 mmol) of PPh₃, 110 mg (0.19 mmol) of Pd(dba)₂, 110 mg (0.57mmol) of CuI and 0.75 g (3.2 mmol) of Y4 (one unit wire). The flask wasflushed with argon and then 65 ml of dry DMF was introduced, followed by25 ml of diisopropylamine. The mixture was stirred at 55° C. for 2.5 h.All tsolvents were removed under reduced pressure. The residue wasdissolved in 100 ml of CH₂Cl₂, and the solution was thoroughly washedwith the saturated EDTA solution (2×100 mL). The Removal of CH₂Cl₂ gave2.3 g of crude product. Silica gel (30 gm) was packed with 50%CH₂Cl₂/hexane, upon loading the sample solution, the column was elutedwith 10% ethyl acetate/CH₂Cl₂. The concentration of the fractionscontaining the desired product gave 1.35 gm (2.94 mmol) of the titleproduct, which was further purified by recrystallization from hot hexanesolution as colorless crystals.

Example 5 Synthesis of Nucleoside Attached to an Insulator

As shown in FIG. 13.

Synthesis of C108: To a flask was added 2.0 gm (3.67 mmol) of2′-amino-5′-O-DMT uridine, 1.63 gm (3.81 mmol) of C44, 5 ml of TEA and100 ml of dichloromethane: This reaction mixture was stirred at roomtemperature over for 72 hrs. The solvent was removed and dissolved in asmall volume of CH₂Cl₂. Silica gel (35 gm) was packed with 2% CH₃OH/1%TEA/CH₂Cl₂, upon loading the sample solution, the column was eluted withthe same solvent system. The fractions containing the desired productwere pooled and concentrated to afford 2.5 gm (80.4%) of the titleproduct.

Synthesis of C109: To a flask was added 2.4 gm (2.80 mmol) of C108, 4 mlof diisopropylethylamine and 80 ml of CH₂Cl₂ under presence of argon.The reaction mixture was cooled in an ice-water bath. Once cooled, 2.10gm (8.83 mmol) of 2-cyanoethyl diisopropylchloro-phosphoramidite wasadded. The mixture was then stirred overnight. The reaction mixture wasdiluted by adding 10 ml of methanol and 150 ml of CH₂Cl₂. This mixturewas washed with a 5% NaHCO₃ solution, dried over sodium sulfate and thenconcentrated for column chromatography. A 65 gm-silica gel column waspacked in 1% TEA and Hexane. The crude product was loaded and the columnwas eluted with 1% TEA/0-20 CH₂Cl₂/Hexane. The fractions containing thedesired product were pooled and concentrated to afford 2.69 gm (90.9%)of the title product.

Example 6 Synthesis of an Electrode Containing Capture Nucleic AcidsContaining Conductive Oligomers and Insulators

Using the above techniques, and standard nucleic acid synthesis, captureprobes comprising a conductive oligomer were made (hereinafter“wire-1”). Conductive oligomers with acetylene termini were made asoutlined herein (hereinafter “wire-2”).

HS—(CH2)16—OH (herein “insulator-2”) was made as follows.

16-Bromohexadecanoic acid: 16-Bromohexadecanoic acid was prepared byrefluxing for 48 hrs 5.0 gr (18.35 mmole) of 16-hydroxyhexadecanoic acidin 24 ml of 1:1 v/v mixture of HBr (48% aqueous solution) and glacialacetic acid. Upon cooling, crude product was solidified inside thereaction vessel. It was filtered out and washed with 3×100 ml of coldwater. Material was purified by recrystallization from n-hexane,filtered out and dried on high vacuum. 6.1 gr (99% yield) of the desiredproduct were obtained.

16-Mercaptohexadecanoic acid. Under inert atmosphere 2.0 gr of sodiummetal suspension (40% in mineral oil) were slowly added to 100 ml of drymethanol at 0° C. At the end of the addition reaction mixture wasstirred for 10 min at RT and 1.75 ml (21.58 mmole) of thioacetic acidwere added. After additional 10 min of stirring, 30 ml degassedmethanolic solution of 6.1 gr (18.19 mmole) of 16bromohexadecanoic acidwere added. The resulted mixture was refluxed for 15 hrs, after which,allowed to cool to RT and 50 ml of degassed 1.0 M NaOH aqueous solutionwere injected. Additional refluxing for 3 hrs required for reactioncompletion. Resulted reaction mixture was cooled with ice bath andpoured, with stirring, into a vessel containing 200 ml of ice water.This mixture was titrated to pH=7 by 1.0 M HCl and extracted with 300 mlof ether. The organic layer was separated, washed with 3×150 ml ofwater, 150 ml of saturated NaCI aqueous solution and dried over sodiumsulfate. After removal of ether material was purified byrecrystallization from n-hexane, filtering out and drying over highvacuum. 5.1 gr (97% yield) of the desired product was obtained.

16-Bromohexadecan-l-ol. Under inert atmosphere 10 ml of BH₃ THF complex(1.0 M THF solution) were added to 30 ml THF solution of 2.15 gr (6.41mmole) of 16-bromohexadecanoic acid at −20° C. Reaction mixture wasstirred at this temperature for 2 hrs and then additional 1 hr at RT.After that time the resulted mixture was poured, with stirring, into avessel containing 200 ml of ice/saturated sodium bicarbonate aqueoussolution. Organic compounds were extracted with 3×200 ml of ether. Theether fractions were combined and dried over sodium sulfate. Afterremoval of ether material was dissolved in minimum amount ofdichloromethane and purified by silica gel chromatography (100%dichloromethane as eluent). 1.92 gr (93% yield) of the desired productwere obtained.

16-Mercaptohexadecan-l-ol. Under inert atmosphere 365 mg of sodium metalsuspension (40% in mineral oil) were added dropwise to 20 ml of drymethanol at 0° C. After completion of addition the reaction mixture wasstirred for 10 min at RT followed by addition of 0.45 ml (6.30 mmole) ofthioacetic acid. After additional 10 min of stirring 3 ml degassedmethanolic solution of 1.0 gr (3.11 mmole) of 16-bromohexadecan-l-olwere added. The resulted mixture was refluxed for 15 hrs, allowed tocool to RT and 20 ml of degassed 1.0 M NaOH aqueous solution wereinjected. The reaction completion required additional 3 hr of reflux.Resulted reaction mixture was cooled with ice bath and poured, withstirring, into a vessel containing 200 ml of ice water. This mixture wastitrated to pH=7 by 1.0 M HCl and extracted with 300 ml of ether. Theorganic layer was separated, washed with 3×150 ml of water, 150 ml ofsaturated NaCl aqueous solution and dried over sodium sulfate. Afterether removal material was dissolved in minimum amount ofdichloromethane and purified by silica gel chromatography (100%dichloromethane as eluent). 600 mg (70% yield) of the desired productwere obtained.

A clean gold covered microscope slide was incubated in a solutioncontaining 100 micromolar HS—(CH₂)₁₆—COOH in ethanol at room temperaturefor 4 hours. The electrode was then rinsed thoroughly with ethanol anddried. 20-30 microliters of wire-1+wire-2 solution (1 micromolar in1×SSC buffer at pH 7.5) was applied to the electrode in a round droplet.The electrode was incubated at room temperature for 4 hours in a moistchamber to minimize evaporation. The wire-1 solution was then removedfrom the electrode and the electrode was immersed in 1×SSC bufferfollowed by 4 rinses with 1×SSC. The electrode was then stored at roomtemperature for up to 2 days in 1×SSC.

Alternatively, and preferably, either a “two-step” or “three-step”process is used. The “two-step” procedure is as follows. The wire 1+wire−2 mixture, in water at ˜5-10 micromolar concentration, was exposed to aclean gold surface and incubated for ˜24 hrs. It was rinsed well withwater and then ethanol. The gold was then exposed to a solution of −100micromolar insulator thiol in ethanol for ˜12 hrs, and rinsed well.Hybridization was done with complement for over 3 hrs. Generally, thehybridization solution was warmed to 50° C., then cooled in order toenhance hybridization.

The “three-step” procedure uses the same concentrations and solvents asabove. The clean gold electrode was incubated in insulator solution for˜1 hr and rinsed. This procedure presumably results in an incompletemonolayer, which has areas of unreacted gold. The slide was thenincubated with a mixture of wire-1 and wire-2 solution for over 24 hrs(generally, the longer the better). These wires still had theethyl-pyridine protecting group on it. The wire solution was 5% NH4OH,15% ethanol in water. This removed the protecting group from the wireand allowed it to bind to the gold (an in situ deprotection). The slidewas then incubated in insulator again for −12 hrs, and hybridized asabove.

In general, a variety of solvent can be used including water, ethanol,acetonitrile, buffer, mixtures etc. Also, the input of energy such asheat or sonication appears to speed up all of the deposition processes,although it may not be necessary. Also, it seems that longer incubationperiods for both steps, for example as long as a week, the better theresults.

Example 7 AC Detection Methods

Electrodes containing the different compositions of the invention weremade and used in AC detection methods. The experiments were run asfollows. A DC offset voltage between the working (sample) electrode andthe reference electrode was swept through the electrochemical potentialof the ferrocene, typically from 0 to 500 mV. On top of the DC offset,an AC signal of variable amplitude and frequency was applied. The ACcurrent at the excitation frequency was plotted versus the DC offset.

Example 8 Comparison of Different ETM Attachments

A variety of different ETM attachments as depicted in FIG. 15 werecompared. As shown in Table 1, a detection probe was attached to theelectrode surface (the sequence containing the wire in the table).Positive (i.e. probes complementary to the detection probe) and negative(i.e. probes not complementary to the detection probe) control labelprobes were added.

Electrodes containing the different compositions of the invention weremade and used in AC detection methods. The experiments were run asfollows. A DC offset voltage between the working (sample) electrode andthe reference electrode was swept through the electrochemical potentialof the ferrocene, typically from 0 to 500 mV. On top of the DC offset,an AC signal of variable amplitude and frequency was applied. The ACcurrent at the excitation frequency was plotted versus the DC offset.

The results are shown in Table 2, with the Y63, VI and IV compoundsshowing the best results.

Redox Metal Potential Complexes (mV) 10 Hz 100 Hz 1,000 Hz 10,000 Hz 1400 Not clear Not clear Not clear Not clear II 350 0.15 μA 0.01 μA 0.005μA ND III 360 0.025 μA 0.085 μA 0.034 μA ND (+control) III 360 0.022 μA0.080 μA 0.090 μA ND (−control) IV 140 0.34 μA 3.0 μA 13.0 μA 35 μA V400 0.02 μA ND 0.15 μA ND VI(1) 140 0.22 μA 1.4 μA 4.4 μA 8.8 μA VI(2)140 0.22 μA 0.78 μA 5.1 μA 44 μA VII 320 0.04 μA ND 0.45 μA No PeakVIII(not 360 0.047 μA ND ND No Peak purified) Y63 160 .25 μA ND 36 μA130 μA Not clear: There is no difference between positive control andnegative control. ND: Not determined

Table of the Oligonucleotides Containing Different Metal ComplexesPositive Control Negative Control Metal Sequence Containing MetalSequence Containing Metal Complexes Complexes and Numbering Complexesand Numbering 1 5′-A(I)C (I)GA GTC CAT GGT-3′ 5′-A(I)G (I)CC TAG CTGGTG-3′ #D199_1 #D200_1 (SEQ ID NO:28) (SEQ ID NO:29) II 5′-A(II)C (II)GAGTC CAT GGT-3′ 5′-A(II)G (II)CC TAG CTG GTG-3′ #D211_1, 2 #D212_1 (SEQID NO:30) (SEQ ID NO:31) III 5′-AAC AGA GTC CAT GGT-3′ 5′-ATG TCC TAGCTG GTG-3′ #D214_1 #D57_1 (SEQ ID NO:32) (SEQ ID NO:33) IV 5′-A(IV)C(IV)GA GTC CAT GGT-3′ 5′-A(IV)G (IV)CC TAG CTG GTG-3′ #D215_1 #D216_1(SEQ ID NO:34) (SEQ ID NO:35) V 5′-A(V)C (V)GA GTC CAT GGT-3′ 5′-A(V)G(V)CC TAG CTG GTG-3′ #D203_1 #D204_1 (SEQ ID NO:36) (SEQ ID NO:37) VI5′-A(VI)C AGA GTC CAT GGT-3′ 5′-A(VI)G TCC TAG CTG GTG-3′ #D205_1#D206_1 (SEQ ID NO:38) (SEQ ID NO:39) VI 5′-A(VI)* AGA GTC CAT GGT-3′5′A(VI)* TCC TAG CTG GTG-3′ #D207_1 #D208_1 (SEQ ID NO:40) (SEQ IDNO:41) VII 5′-A(VII)C (VII)GA GTC CAT GGT-3′ 5′-A(VII)G (VII)CC TAG CTGGTG-3′ #D158_3 #D101_2 (SEQ ID NO:42) (SEQ ID NO:43) VIII 5′-A(VIII)C(VIII)GA GTC CAT GGT-3′ 5′-A(VIII)G (VIII)CC TAG CTG GTG-3′ #D217_1, 2,3 #D218_1 (SEQ ID NO:44) (SEQ ID NO:45) Metal Sequence Containing WireOn G Complexes Surface and Numbering 1 5′-ACC ATG GAC TCT GT(Uw)-3′#D201_1, 2 (SEQ ID NO:46) II 5′-ACC ATG GAC TCT GT(Uw)-3′ #D201_1, 2(SEQ ID NO:46) III 5′-ACC ATG GAC TCT GT(Uw)3′ #D201_1, 2 (SEQ ID NO:46)IV 5′-ACC ATG GAC TCT GT(Uw)3′ #D201_1, 2 (SEQ ID NO:46) V 5′-ACC ATGGAC TCA GA(Uw)-3′ #D83_17, 18 (SEQ ID NO:47) VI 5′-ACC ATG GAC TCTGT(Uw)-3′ #D201_1, 2 (SEQ ID NO:46) VI 5′-ACC ATG GAC TCT GT(Uw)-3′#D201_1, 2 (SEQ ID NO:46) VII 5′-ACC ATG GAC TCA GA(Uw)3′ #D83_17, 18(SEQ ID NO:47) VIII 5′-ACC ATG GAC TCA GA(Uw)-3′ #D83_17, 18 (SEQ IDNO:47)

Example 9 Preferred Embodiments of the Invention

A variety of systems have been run and shown to work well, as outlinedbelow. All compounds are referenced in the Figures. Generally, thesystems were run as follows. The surfaces were made, comprising theelectrode, the capture probe attached via an attachment linker, theconductive oligomers, and the insulators, as outlined above. The othercomponents of the system, including the target sequences, the captureextender probes, and the label probes, were mixed and generally annealedat 90° C. for 5 minutes, and allowed to cool to room temperature for anhour. The mixtures were then added to the electrodes, and AC detectionwas done.

Use of a capture probe, a capture extender probe, an unlabeled targetsequence and a label probe:

A capture probe D112 (SEQ ID NO:7), comprising a 25 base sequence, wasmixed with the Y5 conductive oligomer and the M44 insulator at a ratioof 2:2:1 using the methods above. A capture extender probe D179 (SEQ IDNO:1), comprising a 24 base sequence perfectly complementary to the D112(SEQ ID NO:7) capture probe, and a 24 base sequence perfectlycomplementary to the 2tar (SEQ ID NO:10) target, separated by a singlebase, was added, with the 2tar (SEQ ID NO:10) target. The D 179 (SEQ IDNO:1) molecule carries a ferrocene (using a C15 linkage to the base) atthe end that is closest to the electrode. When the attachment linkersare conductive oligomers, the use of an ETM at or near this positionallows verification that the D179 (SEQ ID NO:1) molecule is present.ferrocene at this position has a different redox potential than the ETMsused for detection. A label probe D309 (SEQ ID NO:2) (dendrimer) wasadded, comprising a 18 base sequence perfectly complementary to aportion of the target sequence, a 13 base sequence linker and fourferrocenes attached using a branching configuration. A representativescan is shown in FIG. 16A. When the 2tar (SEQ ID NO:10) target was notadded, a representative scan is shown in FIG. 16B. No furtherrepresentative scans are shown.

Use of a capture probe and a labeled target sequence:

Example A: A capture probe D94 (SEQ ID NO:8) was added with the Y5 andM44 conductive oligomer at a 2:2:1 ratio with the total thiolconcentration being 833 μM on the electrode surface, as outlined above.A target sequence (D336) (SEQ ID NO:25) comprising a 15 base sequenceperfectly complementary to the D94 (SEQ ID NO:8) capture probe, a 14base linker sequence, and 6 ferrocenes linked via the N6 compound wasused. A representative scan is shown in FIG. 20C. The use of a differentcapture probe, D109 (SEQ ID NO:9), that does not have homology with thetarget sequence, served as the negative control.

Example B: A capture probe D94 (SEQ ID NO:8) was added with the Y5 andM44 conductive oligomer at a 2:2:1 ratio with the total thiolconcentration being 833 μM on the electrode surface, as outlined above.A target sequence (D429) (SEQ ID NO:27) comprising a 15 base sequenceperfectly complementary to the D94 (SEQ ID NO:8) capture probe, a C131ethylene glycol linker hooked to 6 ferrocenes linked via the N6 compoundwas used. A representative scan is shown in FIG. 20E. The use of adifferent capture probe, D109 (SEQ ID NO:9), that does not have homologywith the target sequence, served as the negative control.

Use of a capture probe, a capture extender probe, an unlabeled targetsequence and two label probes with long linkers between the targetbinding sequence and the ETMs:

The capture probe D112 (SEQ ID NO:7), Y5 conductive oligomer, the M44insulator, and capture extender probe D179 (SEQ ID NO:1) were asoutlined above. Two label probes were added: D295 (SEQ ID NO:3)comprising an 18 base sequence perfectly complementary to a portion ofthe target sequence, a 15 base sequence linker and six ferrocenesattached using the N6 linkage depicted in the Figures. D297 (SEQ IDNO:4) is the same, except that it's 18 base sequence hybridizes to adifferent portion of the target sequence.

Use of a capture probe, a capture extender probe, an unlabeled targetsequence and two label probes with short linkers between the targetbinding sequence and the ETMs;

The capture probe D112 (SEQ ID NO:7), Y5 conductive oligomer, the M44insulator, and capture extender probe D179 (SEQ ID NO:1) were asoutlined above. Two label probes were added: D296 (SEQ ID NO:6)comprising an 18 base sequence perfectly complementary to a portion ofthe target sequence, a 5 base sequence linker and six ferrocenesattached using the N6 linkage depicted in FIG. 23. D298 (SEQ ID NO:5) isthe same, except that it's 18 base sequence hybridizes to a differentportion of the target sequence.

Use of two capture probes, two capture capture extender probes, anunlabeled large target sequence and two label probes with long linkersbetween the target binding sequence and the ETMs;

This test was directed to the detection of rRNA. The Y5 conductiveoligomer, the M44 insulator, and one surface probe D350 that wascomplementary to 2 capture sequences D417 (SEQ ID NO:16) and EU1 (SEQ IDNO:17) were used as outlined herein. The D350, Y5 and M44 was added at a0.5:4.5:1 ratio. Two capture extender probes were used; D417 (SEQ IDNO:16) that has 16 bases complementary to the D350 capture probe and 21bases complementary to the target sequence, and EU1 (SEQ ID NO:17) thathas 16 bases complementary to the D350 capture probe and 23 basescomplementary to a different portion of the target sequence. Two labelprobes were added: D468 (SEQ ID NO:14) comprising a 30 base sequenceperfectly complementary to a portion of the target sequence, a linkercomprising three glen linkers as shown in FIG. 15 (comprisingpolyethylene glycol) and six ferrocenes attached using N6. D449 (SEQ IDNO:15) is the same, except that it's 28 base sequence hybridizes to adifferent portion of the target sequence, and the polyethylene glycollinker used (C131) is shorter.

Use of a capture probe, an unlabeled target, and a label probe:

Example A: A capture probe D112 (SEQ ID NO:7), Y5 conductive oligomerand the M44 insulator were put on the electrode at 2:2:1 ratio with thetotal thiol concentration being 833 μM. A target sequence MT1 (SEQ IDNO:18) was added, that comprises a sequence complementary to D112 (SEQID NO:7) and a 20 base sequence complementary to the label probe D358(SEQ ID NO:19) were combined; in this case, the label probe D358 (SEQ IDNO:19) was added to the target sequence prior to the introduction to theelectrode. The label probe contains six ferrocenes attached using the N6linkages depicted in the Figures. The replacement of MT1 (SEQ ID NO:18)with NC112 (SEQ ID NO:24) which is not complementary to the captureprobe resulted in no signal; similarly, the removal of MT1 (SEQ IDNO:18) resulted in no signal.

Example B. A capture probe D334 (SEQ ID NO:20), Y5 conductive oligomerand the M44 insulator were put on the electrode at 2:2:1 ratio with thetotal thiol concentration being 833 μM. A target sequence LP280 (SEQ IDNO:22) was added, that comprises a sequence complementary to the captureprobe and a 20 base sequence complementary to the label probe D335 (SEQID NO:21) were combined; in this case, the label probe D335 (SEQ IDNO:21) was added to the target prior to introduction to the electrode.The label probe contains six ferrocenes attached using the N6 linkagesdepicted in the Figures. Replacing LP280 (SEQ ID NO:22) with the LN280(SEQ ID NO:23) probe (which is complementary to the label probe but notthe capture probe) resulted in no signal.

1. A method of detecting a target analyte in a sample comprising: a)contacting said sample with an electrode comprising: i) a monolayercomprising insulators; and ii) a capture binding ligand wherein saidcapture binding ligand is covalently attached to said electrode via anattachment linker, under conditions wherein said target analyte binds tosaid capture binding ligand; b) binding a target analyte binding ligandto said target analyte, wherein said target analyte binding ligandcomprises at least one non-electrochemiluminescent electron transfermoiety (ETM) covalently attached to said target analyte binding ligand;and c) detecting the presence of said target analyte.
 2. A methodaccording to claim 1 wherein said attachment linker is selected from thegroup consisting of conductive oligomers and insulators.
 3. A methodaccording to claim 1 wherein said capture binding ligand is a nucleicacid.
 4. A method according to claim 1 wherein said capture bindingligand is a protein.
 5. A method according to claim 1 wherein saidtarget analyte is a protein.
 6. A method according to claim 1 whereinsaid target analyte is a peptide.
 7. A method according to claim 1wherein said target analyte is a nucleic acid.
 8. A method according toclaim 1 wherein said ETM is a metallocene.
 9. A method according toclaim 8 wherein said metallocene is ferrocene.
 10. A method according toclaim 1 wherein said insulators are alkyl chains.
 11. A method ofdetecting a target analyte in a sample comprising: a) contacting saidsample with an electrode comprising: i) a monolayer comprisinginsulators; and ii) a capture binding ligand wherein said capturebinding ligand is covalently attached to said electrode via anattachment linker, under conditions wherein said target analyte binds tosaid capture binding ligand; b) binding a target analyte binging ligandto said target analyte, wherein said target analyte binding ligandcomprises: i) nucleic acid; ii) at least one non-electrochemiluminescentelectron transfer moiety (ETM) covalently attached to said targetanalyte binding ligand; and c) detecting the presence of said targetanalyte.
 12. A method according to claim 11 wherein said attachmentlinker is selected from the group consisting of conductive oligomers andinsulators.
 13. A method according to claim 11 wherein said capturebinding ligand is a nucleic acid.
 14. A method according to claim 11wherein said capture binding ligand is a protein.
 15. A method accordingto claim 11 wherein said ETM is a metallocene.
 16. A method according toclaim 15 wherein said metallocene is ferrocene.
 17. A method accordingto claim 11 wherein said insulators are alkyl chains.
 18. A methodaccording to claim 1 wherein said monolayer further comprises conductiveoligomers.
 19. A method according to claim 11 wherein said monolayerfurther comprises conductive oligomers.
 20. A method according to claim2 wherein said attachment linker is a conductive oligomer.
 21. A methodaccording to claim 2 wherein said attachment linker is an insulator. 22.A method according to claim 2 or 12 wherein said attachment linker is aconductive oligomer.
 23. A method according to claim 2 or 12 whereinsaid attachment liker is an insulator.
 24. A method of detecting atarget analyte in a sample comprising: a) contacting said sample with anelectrode comprising: i) a monolayer comprising insulators; and ii) acapture binding ligand wherein said capture binding ligand is covalentlyattached to said electrode via an attachment linker, under conditionswherein said target analyte binds to said capture binding ligand; b)binding a target analyte binding ligand to said target analyte, whereinsaid target analyte binding ligand comprising at least one electrontransfer moiety (ETM) covalently attached to said target analyte bindingligand; and c) detecting the presence of said target analyteelectronically.